RNA Interference Mediated Inhibition Of Gene Expression Using Multifunctional Short Interfering Nucleic Acid (Multifunctional siNA)

ABSTRACT

The present invention concerns methods and nucleic acid based reagents useful in modulating gene expression in a variety of applications, including use in therapeutic, veterinary, agricultural, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to multifunctional short interfering nucleic acid (multifunctional siNA) molecules that modulate the expression of one or more genes in a biologic system, such as a cell, tissue, or organism via RNA interference (RNAi). The bifunctional short interfering nucleic acid (multifunctional siNA) molecules of the invention can target more than one regions of nucleic acid sequence in a single target nucleic acid molecule or can target regions of nucleic acid sequence in differing target nucleic acid molecules. The self multifunctional siNA molecules are useful in the treatment of any disease or condition that responds to modulation of gene expression or activity in a cell, tissue, or organism.

This application is a division of application Ser. No. 10/597,755 filedAug. 7, 2006, which is a national stage of PCT/US05/004270 filed Feb. 9,2005, which claims the benefit of 60/543,480 filed Feb. 10, 2004, andPCT/US05/004270 filed Feb. 9, 2005 is a continuation-in-part ofApplication No. PCT/US04/16390 filed May 24, 2004. All of theseapplications are incorporated by reference in their entirety.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR§1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “SequenceListing4USDIV,”created on Nov. 16, 2010, which is 49,405 bytes in size.

FIELD OF THE INVENTION

The present invention concerns methods and reagents useful in modulatinggene expression in a variety of applications, including use intherapeutic, veterinary, agricultural, diagnostic, target validation,and genomic discovery applications. Specifically, the invention relatesto multifunctional short interfering nucleic acid (multifunctional siNA)molecules that modulate the expression of more than one gene and methodsof generating such siNA molecules.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to nucleic acidmolecules that moduate gene expression. The discussion is provided onlyfor understanding of the invention that follows. The summary is not anadmission that any of the work described below is prior art to theclaimed invention.

Various single strand, double strand, and triple strand nucleic acidmolecules are presently known that possess biological activity. Examplesof single strand nucleic acid molecules that have biologic activity tomediate alteration of gene expression include antisense nucleic acidmolecules, enzymatic nucleic acid molecules or ribozymes, and2′-5′-oligoadenylate nucleic acid molecules. Examples of triple strandnucleic acid molecules that have biologic activity to mediate alterationof gene expression include triplex forming oligonucleotides. Examples ofdouble strand nucleic acid molecules that have biologic activity tomediate alteration of gene expression include dsRNA and siRNA. Forexample, interferon mediated induction of protein kinase PKR is known tobe activated in a non-sequence specific manner by long double strandedRNA (see for example Wu and Kaufman, 1997, J. Biol. Chem., 272, 1921-6).This pathway shares a common feature with the 2′,5′-linkedoligoadenylate (2-5A) system in mediating RNA cleavage via RNaseL (seefor example Cole et al., 1997, J. Biol. Chem., 272, 19187-92). Whereasthese responses are intrinsically sequence-non-specific, inhibition ofgene expression via short interfering RNA mediated RNA interference(RNAi) is known to be highly sequence specific (see for example Elbashiret al., 2001, Nature, 411, 494-498).

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951). The corresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA or viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response though amechanism that has yet to be fully characterized. This mechanism appearsto be different from the interferon response that results fromdsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylatesynthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Hamilton et al., supra; Zamore et al., 2000,Cell, 101, 25-33; Berstein et al., 2001, Nature, 409, 363). Shortinterfering RNAs derived from dicer activity are typically about 21 toabout 23 nucleotides in length and comprise about 19 base pair duplexes(Hamilton et al., supra; Elbashir et al., 2001, Genes Dev., 15, 188).Dicer has also been implicated in the excision of 21- and 22-nucleotidesmall temporal RNAs (stRNAs) from precursor RNA of conserved structurethat are implicated in translational control (Hutvagner et al., 2001,Science, 293, 834). The RNAi response also features an endonucleasecomplex, commonly referred to as an RNA-induced silencing complex(RISC), which mediates cleavage of single-stranded RNA having sequencecomplementary to the antisense strand of the siRNA duplex. Cleavage ofthe target RNA takes place in the middle of the region complementary tothe antisense strand of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., InternationalPCT Publication No. WO 01/75164, describe RNAi induced by introductionof duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cellsincluding human embryonic kidney and HeLa cells. Recent work inDrosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877and Tuschl et al., International PCT Publication No. WO 01/75164) hasrevealed certain requirements for siRNA length, structure, chemicalcomposition, and sequence that are essential to mediate efficient RNAiactivity. These studies have shown that 21-nucleotide siRNA duplexes aremost active when containing 3′-terminal dinucleotide overhangs.Furthermore, complete substitution of one or both siRNA strands with2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity,whereas substitution of the 3′-terminal siRNA overhang nucleotides with2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatchsequences in the center of the siRNA duplex were also shown to abolishRNAi activity. In addition, these studies also indicate that theposition of the cleavage site in the target RNA is defined by the 5′-endof the siRNA guide sequence rather than the 3′-end of the guide sequence(Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicatedthat a 5′-phosphate on the target-complementary strand of a siRNA duplexis required for siRNA activity and that ATP is utilized to maintain the5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al.,International PCT Publication No. WO 01/75164). In addition, Elbashir etal., supra, also report that substitution of siRNA with 2′-O-methylnucleotides completely abolishes RNAi activity. Li et al., InternationalPCT Publication No. WO 00/44914, and Beach et al., International PCTPublication No. WO 01/68836 preliminarily suggest that siRNA may includemodifications to either the phosphate-sugar backbone or the nucleosideto include at least one of a nitrogen or sulfur heteroatom, however,neither application postulates to what extent such modifications wouldbe tolerated in siRNA molecules, nor provides any further guidance orexamples of such modified siRNA. Kreutzer et al., Canadian PatentApplication No. 2,359,180, also describe certain chemical modificationsfor use in dsRNA constructs in order to counteract activation ofdouble-stranded RNA-dependent protein kinase PKR, specifically 2′-aminoor 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer et al. similarly fails to provideexamples or guidance as to what extent these modifications would betolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et al., International PCT Publication No. WO 01/75164,describe a Drosophila in vitro RNAi system and the use of specific siRNAmolecules for certain functional genomic and certain therapeuticapplications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubtsthat RNAi can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. WO 00/44914, describe the use of specific long (141bp-488 bp) enzymatically synthesized or vector expressed dsRNAs forattenuating the expression of certain target genes. Zernicka-Goetz etal., International PCT Publication No. WO 01/36646, describe certainmethods for inhibiting the expression of particular genes in mammaliancells using certain long (550 bp-714 bp), enzymatically synthesized orvector expressed dsRNA molecules. Fire et al., International PCTPublication No. WO 99/32619, describe particular methods for introducingcertain long dsRNA molecules into cells for use in inhibiting geneexpression in nematodes. Plaetinck et al., International PCT PublicationNo. WO 00/01846, describe certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific long dsRNA molecules. Mello et al., International PCTPublication No. WO 01/29058, describe the identification of specificgenes involved in dsRNA-mediated RNAi. Deschamps Depaillette et al.,International PCT Publication No. WO 99/07409, describe specificcompositions consisting of particular dsRNA molecules combined withcertain anti-viral agents. Waterhouse et al., International PCTPublication No. 99/53050, describe certain methods for decreasing thephenotypic expression of a nucleic acid in plant cells using certaindsRNAs. Driscoll et al., International PCT Publication No. WO 01/49844,describe specific DNA expression constructs for use in facilitating genesilencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describespecific chemically-modified dsRNA constructs targeting the unc-22 geneof C. elegans. Grossniklaus, International PCT Publication No. WO01/38551, describes certain methods for regulating polycomb geneexpression in plants using certain dsRNAs. Churikov et al.,International PCT Publication No. WO 01/42443, describe certain methodsfor modifying genetic characteristics of an organism using certaindsRNAs. Cogoni et al., International PCT Publication No. WO 01/53475,describe certain methods for isolating a Neurospora silencing gene anduses thereof. Reed et al., International PCT Publication No. WO01/68836, describe certain methods for gene silencing in plants. Honeret al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophila-derived geneproducts that may be related to RNAi in Drosophila. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/63364, and Satishchandran et al., InternationalPCT Publication No. WO 01/04313, describe certain methods andcompositions for inhibiting the function of certain polynucleotidesequences using certain long (over 250 bp), vector expressed dsRNAs.Echeverri et al., International PCT Publication No. WO 02/38805,describe certain C. elegans genes identified via RNAi. Kreutzer et al.,International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP1144623 B1 describes certain methods for inhibiting gene expressionusing dsRNA. Graham et al., International PCT Publications Nos. WO99/49029 and WO 01/70949, and AU 4037501 describe certain vectorexpressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559,describe certain methods for inhibiting gene expression in vitro usingcertain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi.Martinez et al., 2002, Cell, 110, 563-574, describe certain singlestranded siRNA constructs, including certain 5′-phosphorylated singlestranded siRNAs that mediate RNA interference in Hela cells. All ofthese references describe double stranded nucleic acid constructs whereone of the two strands (the antisense strand) is complementary to thetarget RNA and the other strand (sense strand) is complementary to theantisense strand.

SUMMARY OF THE INVENTION

This invention relates to nucleic acid-based compounds, compositions,and methods useful for modulating RNA function and/or gene expression ina cell. Specifically, the instant invention features multifunctionalshort interfering nucleic acid (multifunctional siNA) molecules thatmodulate the expression of one or more genes in a biologic system, suchas a cell, tissue, or organism. The multifunctional short interferingnucleic acid (multifunctional siNA) molecules of the invention cantarget more than one region of the target nucleic acid sequence or cantarget sequences of more than one distinct target nucleic acidmolecules. The multifunctional siNA molecules of the invention can bechemically synthesized or expressed from transcription units and/orvectors. The multifunctional siNA molecules of the instant inventionprovide useful reagents and methods for a variety of therapeutic,diagnostic, agricultural, veterinary, target validation, genomicdiscovery, genetic engineering and pharmacogenomic applications.

Applicant demonstrates herein that certain oligonucleotides, referred toherein for convenience but not limitation as multifunctional shortinterfering nucleic acid or multifunctional siNA molecules, are potentmediators of sequence specific regulation of gene expression. Themultifunctional siNA molecules of the invention are distinct from othernucleic acid sequences known in the art (e.g., siRNA, miRNA, stRNA,shRNA, antisense oligonucleotides, etc.) in that they represent a classof polynucleotide molecules that are designed such that each strand inthe multifunctional siNA construct comprises nucleotide sequence that iscomplementary to a distinct nucleic acid sequence in one or more targetnucleic acid molecules. A single multifunctional siNA molecule of theinvention can thus target more than one (e.g., 2, 3, 4, 5, or more)differing target nucleic acid target molecules. Nucleic acid moleculesof the invention can also target more than one (e.g., 2, 3, 4, 5, ormore) region of the same target nucleic acid sequence. As suchmultifunctional siNA molecules of the invention are useful in downregulating or inhibiting the expression of one or more target nucleicacid molecules. For example, a multifunctional siNA molecule of theinvention can target nucleic acid molecules encoding a cytokine and itscorresponding receptor(s), nucleic acid molecules encoding a virus orviral proteins and corresponding cellular proteins required for viralinfection and/or replication, or differing strains of a particularvirus. By reducing or inhibiting expression of more than one targetnucleic acid molecule with one multifunctional siNA construct,multifunctional siNA molecules of the invention represent a class ofpotent therapeutic agents that can provide simultaneous inhibition ofmultiple targets within a disease related pathway. Such simultaneousinhibition can provide synergistic therapeutic treatment strategieswithout the need for separate preclinical and clinical developmentefforts or complex regulatory approval process.

Use of multifunctional siNA molecules that target more then one regionof a target nucleic acid molecule (e.g., messenger RNA) is expected toprovide potent inhibition of gene expression. For example, a singlemultifunctional siNA construct of the invention can target bothconserved and variable regions of a target nucleic acid molecule,thereby allowing down regulation or inhibition of different splicevariants encoded by a single gene, or allowing for targeting of bothcoding and non-coding regions of a target nucleic acid molecule.

Generally, double stranded oligonucleotides are formed by the assemblyof two distinct oligonucleotide sequences where the oligonucleotidesequence of one strand is complementary to the oligonucleotide sequenceof the second strand; such double stranded oligonucleotides aregenerally assembled from two separate oligonucleotides (e.g., siRNA), orfrom a single molecule that folds on itself to form a double strandedstructure (e.g. shRNA or short hairpin RNA). These double strandedoligonucleotides known in the art all have a common feature in that eachstrand of the duplex has a distinct nucleotide sequence, wherein onlyone nucleotide sequence region (guide sequence or the antisensesequence) has complementarity to a target nucleic acid sequence and theother strand (sense sequence) comprises nucleotide sequence that ishomologous to the target nucleic acid sequence. Generally, the antisensesequence is retained in the active RISC complex and guides the RISC tothe target nucleotide sequence by means of complementary base-pairing ofthe antisense sequence with the target sequence for mediatingsequence-specific RNA interference. It is known in the art that in somecell culture systems, certain types of unmodified siRNAs can exhibit“off target” effects. It is hypothesized that this off-target effectinvolves the participation of the sense sequence instead of theantisense sequence of the siRNA in the RISC complex (see for exampleSchwarz et al., 2003, Cell, 115, 199-208). In this instance the sensesequence is believed to direct the RISC complex to a sequence(off-target sequence) that is distinct from the intended targetsequence, resulting in the inhibition of the off-target sequence. Inthese double stranded nucleic acid molecules, each strand iscomplementary to a distinct target nucleic acid sequence. However, theoff-targets that are affected by these dsRNAs are not entirelypredictable and are non-specific.

Distinct from the double stranded nucleic acid molecules known in theart, the applicants have developed a novel, potentially cost effectiveand simplified method of down regulating or inhibiting the expression ofmore than one target nucleic acid sequence using a singlemultifunctional siNA construct. The multifunctional siNA molecules ofthe invention are designed such that a portion of each strand or regionof the multifunctional siNA is complementary to a target nucleic acidsequence of choice. As such, the multifunctional siNA molecules of theinvention are not limited to targeting sequences that are complementaryto each other, but rather to any two differing target nucleic acidsequences. Multifunctional siNA molecules of the invention are designedsuch that each strand or region of the multifunctional siNA moleculethat is complementary to a given target nucleic acid sequence is oflength suitable (e.g., from about 16 to about 28 nucleotides in length,preferably from about 18 to about 28 nucleotides in length) formediating RNA interference against the target nucleic acid sequence. Themultifunctional siNA of the invention is expected to minimize off-targeteffects seen with certain siRNA sequences, such as those described in(Schwarz et al., supra).

It has been reported that dsRNAs of length between 29 base pairs and 36base pairs (Tuschl et al., International PCT Publication No. WO02/44321) do not mediate RNAi. One reason these dsRNAs are inactive maybe the lack of turnover or dissociation of the strand that interactswith the target RNA sequence, such that the RISC complex is not able toefficiently interact with multiple copies of the target RNA resulting ina significant decrease in the potency and efficiency of the RNAiprocess. Applicant has surprisingly found that the multifunctional siNAsof the invention can overcome this hurdle and are capable of enhancingthe efficiency and potency of RNAi process. As such, in certainembodiments of the invention, multifunctional siNAs of length betweenabout 29 to about 36 base pairs can be designed such that, a portion ofeach strand of the multifunctional siNA molecule comprises a nucleotidesequence region that is complementary to a target nucleic acid of lengthsufficient to mediate RNAi efficiently (e.g., about 15 to about 23 basepairs) and a nucleotide sequence region that is not complementary to thetarget nucleic acid. By having both complementary and non-complementaryportions in each strand of the multifunctional siNA, the multifunctionalsiNA can mediate RNA interference against a target nucleic acid sequencewithout being prohibitive to turnover or dissociation (e.g., where thelength of each strand is too long to mediate RNAi against the respectivetarget nucleic acid sequence). Furthermore, design of multifunctionalsiNA molecules of the invention with internal overlapping regions allowsthe multifunctional siNA molecules to be of favorable (decreased) sizefor mediating RNA interference and of size that is well suited for useas a therapeutic agent (e.g., wherein each strand is independently fromabout 18 to about 28 nucleotides in length). Non-limiting examples areillustrated in the enclosed FIGS. 1-6.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a first region and a second region, where the first region ofthe multifunctional siNA comprises nucleotide sequence complementary toa nucleic acid sequence of a first target nucleic acid molecule, and thesecond region of the multifunctional siNA comprises nucleic acidsequence complementary to a nucleic acid sequence of a second targetnucleic acid molecule. In one embodiment, a multifunctional siNAmolecule of the invention comprises a first region and a second region,where the first region of the multifunctional siNA comprises nucleotidesequence complementary to a nucleic acid sequence of the first region ofa target nucleic acid molecule, and the second region of themultifunctional siNA comprises nucleotide sequence complementary to anucleic acid sequence of a second region of a the target nucleic acidmolecule. In another embodiment, the first region and second region ofthe multifunctional siNA can comprise separate nucleic acid sequencesthat share some degree of complementarity (e.g., from about 1 to about10 complementary nucleotides). In certain embodiments, multifunctionalsiNA constructs comprising separate nucleic acid sequences can bereadily linked post-synthetically by methods and reagents known in theart and such linked constructs are within the scope of the invention.Alternately, the first region and second region of the multifunctionalsiNA can comprise a single nucleic acid sequence having some degree ofself complementarity, such as in a hairpin or stem-loop structure.Non-limiting examples of such double stranded and hairpinmultifunctional short interfering nucleic acid s are illustrated inFIGS. 1 and 2 respectively. These multifunctional short interferingnucleic acids (multifunctional siNAs) can optionally include certainoverlapping nucleotide sequence where such overlapping nucleotidesequence is present in between the first region and the second region ofthe multifunctional siNA (see for example FIGS. 3 and 4).

In one embodiment, the invention features a multifunctional shortinterfering nucleic acid (multifunctional siNA) molecule, wherein eachstrand of the multifunctional siNA independently comprises a firstregion of nucleic acid sequence that is complementary to a distincttarget nucleic acid sequence and the second region of nucleotidesequence that is not complementary to the target sequence. The targetnucleic acid sequence of each strand is in the same target nucleic acidmolecule or different target nucleic acid molecules.

In another embodiment, the multifunctional siNA comprises two strands,where: (a) the first strand comprises a region having sequencecomplementarity to a target nucleic acid sequence (complementaryregion 1) and a region having no sequence complementarity to the targetnucleotide sequence (non-complementary region 1); (b) the second strandof the multifunction siNA comprises a region having sequencecomplementarity to a target nucleic acid sequence that is distinct fromthe target nucleotide sequence complementary to the first strandnucleotide sequence (complementary region 2), and a region having nosequence complementarity to the target nucleotide sequence ofcomplementary region 2 (non-complementary region 2); (c) thecomplementary region 1 of the first strand comprises nucleotide sequencethat is complementary to nucleotide sequence in the non-complementaryregion 2 of the second strand and the complementary region 2 of thesecond strand comprises nucleotide sequence that is complementary tonucleotide sequence in the non-complementary region 1 of the firststrand. The target nucleic acid sequence of complementary region 1 andcomplementary region 2 is in the same target nucleic acid molecule ordifferent target nucleic acid molecules.

In another embodiment, the multifunctional siNA comprises two strands,where: (a) the first strand comprises a region having sequencecomplementarity to a target nucleic acid sequence derived from a gene(e.g., mammalian gene, viral gene or genome, bacterial gene or a plantgene) (complementary region 1) and a region having no sequencecomplementarity to the target nucleotide sequence of complementaryregion 1 (non-complementary region 1); (b) the second strand of themultifunction siNA comprises a region having sequence complementarity toa target nucleic acid sequence derived from a gene that is distinct fromthe gene of complementary region 1 (complementary region 2), and aregion having no sequence complementarity to the target nucleotidesequence of complementary region 2 (non-complementary region 2); (c) thecomplementary region 1 of the first strand comprises nucleotide sequencethat is complementary to nucleotide sequence in the non-complementaryregion 2 of the second strand and the complementary region 2 of thesecond strand comprises nucleotide sequence that is complementary tonucleotide sequence in the non-complementary region 1 of the firststrand.

In another embodiment, the multifunctional siNA comprises two strands,where: (a) the first strand comprises a region having sequencecomplementarity to target nucleic acid sequence derived from a gene(e.g., mammalian gene, viral gene or genome, bacterial gene or a plantgene) (complementary region 1) and a region having no sequencecomplementarity to the target nucleotide sequence of complementaryregion 1 (non-complementary region 1); (b) the second strand of themultifunction siNA comprises a region having sequence complementarity toa target nucleic acid sequence distinct from the target nucleic acidsequence of complementary region 1(complementary region 2), providedhowever, the target nucleic acid sequence for complementary region 1 andtarget nucleic acid sequence for complementary region 2 are both derivedfrom the same gene, and a region having no sequence complementarity tothe target nucleotide sequence of complementary region 2(non-complementary region 2); (c) the complementary region 1 of thefirst strand comprises nucleotide sequence that is complementary tonucleotide sequence in the non-complementary region 2 of the secondstrand and the complementary region 2 of the second strand comprisesnucleotide sequence that is complementary to nucleotide sequence in thenon-complementary region 1 of the first strand.

In one embodiment, the invention features a multifunctional shortinterfering nucleic acid (multifunctional siNA) molecule, wherein themultifunctional siNA comprises two complementary nucleic acid sequencesin which the first sequence comprises a first region having nucleotidesequence complementary to nucleotide sequence within a target nucleicacid molecule, and in which the second sequence comprises a first regionhaving nucleotide sequence complementary to a distinct nucleotidesequence within the same target nucleic acid molecule. Preferably, thefirst region of the first sequence is also complementary to thenucleotide sequence of the second region of the second sequence, andwhere the first region of the second sequence is complementary to thenucleotide sequence of the second region of the first sequence,

In one embodiment, the invention features a multifunctional shortinterfering nucleic acid (multifunctional siNA) molecule, wherein themultifunctional siNA comprises two complementary nucleic acid sequencesin which the first sequence comprises a first region having nucleotidesequence complementary to nucleotide sequence within a first targetnucleic acid molecule, and in which the second sequence comprises afirst region having nucleotide sequence complementary to a distinctnucleotide sequence within a second target nucleic acid molecule.Preferably, the first region of the first sequence is also complementaryto the nucleotide sequence of the second region of the second sequence,and where the first region of the second sequence is complementary tothe nucleotide sequence of the second region of the first sequence,

In one embodiment, the invention features a multifunctional siNAmolecule comprising a first region and a second region, where the firstregion comprises nucleic acid sequence having between about 18 to about28 nucleotides complementary to a nucleic acid sequence within a firsttarget nucleic acid molecule, and the second region comprises nucleotidesequence having between about 18 to about 28 nucleotides complementaryto a distinct nucleic acid sequence within a second target nucleic acidmolecule.

In one embodiment, the invention features a multifunctional siNAmolecule comprising a first region and a second region, where the firstregion comprises nucleic acid sequence having between about 18 to about28 nucleotides complementary to a nucleic acid sequence within a targetnucleic acid molecule, and the second region comprises nucleotidesequence having between about 18 to about 28 nucleotides complementaryto a distinct nucleic acid sequence within the same target nucleic acidmolecule.

In one embodiment, the invention features a double strandedmultifunctional short interfering nucleic acid (multifunctional siNA)molecule, wherein one strand of the multifunctional siNA comprises afirst region having nucleotide sequence complementary to a first targetnucleic acid sequence, and the second strand comprises a first regionhaving nucleotide sequence complementary to a second target nucleic acidsequence. The first and second target nucleic acid sequences can bepresent in separate target nucleic acid molecules or can be differentregions within the same target nucleic acid molecule. As such,multifunctional siNA molecules of the invention can be used to targetthe expression of different genes, splice variants of the same gene,both mutant and conserved regions of one or more gene transcripts, orboth coding and non-coding sequences of the same or differing genes orgene transcripts.

In one embodiment, a target nucleic acid molecule of the inventionencodes a single protein. In another embodiment, a target nucleic acidmolecule encodes more than one protein (e.g., 1, 2, 3, 4, 5 or moreproteins). As such, a multifunctional siNA construct of the inventioncan be used to down regulate or inhibit the expression of severalproteins. For example, a multifunctional siNA molecule comprising aregion in one strand having nucleotide sequence complementarity to afirst target nucleic acid sequence derived from a gene encoding oneprotein (e.g., a cytokine, such as vascular endothelial growth factor orVEGF) and the second strand comprising a region with nucleotide sequencecomplementarity to a second target nucleic acid sequence present intarget nucleic acid molecules derived from genes encoding two proteins(e.g., two differing receptors, such as VEGF receptor 1 and VEGFreceptor 2, for a single cytokine, such as VEGF) can be used to downregulate, inhibit, or shut down a particular biologic pathway bytargeting, for example, a cytokine and receptors for the cytokine, or aligand and receptors for the ligand.

In one embodiment the invention takes advantage of conserved nucleotidesequences present in different isoforms of cytokines or ligands andreceptors for the cytokines or ligands. By designing multifunctionalsiNAs in a manner where one strand includes sequence that iscomplementary to target nucleic acid sequence conserved among variousisoforms of a cytokine and the other strand includes sequence that iscomplementary to target nucleic acid sequence conserved among thereceptors for the cytokine, it is possible to selectively andeffectively modulate or inhibit a biological pathway or multiple genesin a biological pathway using a single multifunctional siNA.

In another nonlimiting example, a multifunctional siNA moleculecomprising a region in one strand having nucleotide sequencecomplementarity to a first target nucleic acid sequence present intarget nucleic acid molecules encoding two proteins (e.g., two isoformsof a cytokine such as VEGF, including for example any of VEGF-A, VEGF-B,VEGF-C, and/or VEGF-D) and the second strand comprising a region withnucleotide sequence complementarity to a second target nucleic acidsequence present in target nucleotide molecules encoding two additionalproteins (e.g., two differing receptors for the cytokine, such asVEGFR1, VEGFR2, and/or VEGFR3) can be used to down regulate, inhibit, orshut down a particular biologic pathway by targeting different isoformsof a cytokine and receptors for such cytokines.

In another non-limiting example, a multifunctional siNA moleculecomprising a region in one strand having nucleotide sequencecomplementarity to a first target nucleic acid sequence derived from atarget nucleic acid molecule encoding a virus or a viral protein (e.g.,HIV) and the second strand comprising a region having nucleotidesequence complementarity to a second target nucleic acid sequencepresent in target nucleic acid molecule encoding a cellular protein(e.g., a receptor for the virus, such as CCR5 receptor for HIV) can beused to down regulate, inhibit, or shut down the viral replication andinfection by targeting the virus and cellular proteins necessary forviral infection or replication.

In another nonlimiting example, a multifunctional siNA moleculecomprising a region in one strand having nucleotide sequencecomplementarity to a first target nucleic acid sequence (e.g. conservedsequence) present in a target nucleic acid molecule such as a viralgenome (e.g., HIV genomic RNA) and the second strand comprising a regionhaving nucleotide sequence complementarity to a second target nucleicacid sequence (e.g. conserved sequence) present in target nucleic acidmolecule derived from a gene encoding a viral protein (e.g., HIVproteins, such as TAT, REV, ENV or NEF) to down regulate, inhibit, orshut down the viral replication and infection by targeting the viralgenome and viral encoded proteins necessary for viral infection orreplication.

In one embodiment the invention takes advantage of conserved nucleotidesequences present in different strains, isotypes or forms of a virus andgenes encoded by these different strains, isotypes and forms of thevirus. By designing multifunctional siNAs in a manner where one strandincludes sequence that is complementary to target nucleic acid sequenceconserved among various strains, isotypes or forms of a virus and theother strand includes sequence that is complementary to target nucleicacid sequence conserved in a protein encoded by the virus, it ispossible to selectively and effectively inhibit viral replication orinfection using a single multifunctional siNA.

In one embodiment, a multifunctional short interfering nucleic acid(multifunctional siNA) of the invention comprises a region in eachstrand, wherein the region in one strand comprises nucleotide sequencecomplementary to a cytokine and the region in the second strandcomprises nucleotide sequence complementary to a corresponding receptorfor the cytokine. Non-limiting examples of cytokines include vascularendothelial growth factors (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D),interleukins (e.g., IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13), tumor necrosis factors(e.g., TNF-alpha, TNF-beta), colony stimulating factors (e.g., CSFs),interferons (e.g., IFN-gamma), nerve growth factors (e.g., NGFs),epidermal growth factors (e.g., EGF), platelet derived growth factors(e.g., PDGF), fibroblast growth factors (e.g., FGF), transforming growthfactors (e.g., TGF-alpha and TGF-beta), erythropoietins (e.g., Epo), andInsulin like growth factors (e.g., IGF-1, IGF-2) and non-limitingexamples of cytokine receptors include receptors for each of the abovecytokines.

In one embodiment, a multifunctional short interfering nucleic acid(multifunctional siNA) of the invention comprises a first region and asecond region, wherein the first region comprises nucleotide sequencecomplementary to a viral RNA of a first viral strain and the secondregion comprises nucleotide sequence complementary to a viral RNA of asecond viral strain. In one embodiment, the first and second regions cancomprise nucleotide sequence complementary to shared or conserved RNAsequences of differing viral strains or classes or viral strains.Non-limiting examples of viruses include Hepatitis C Virus (HCV),Hepatitis B Virus (HBV), Human Immunodeficiency Virus type 1 (HIV-1),Human Immunodeficiency Virus type 2 (HIV-2), West Nile Virus (WNV),cytomegalovirus (CMV), respiratory syncytial virus (RSV), influenzavirus, rhinovirus, papillomavirus (HPV), Herpes Simplex Virus (HSV),severe acute respiratory virus (SARS), and other viruses such as HTLV.

In one embodiment, a multifunctional short interfering nucleic acid(multifunctional siNA) of the invention comprises a first region and asecond region, wherein the first region comprises nucleotide sequencecomplementary to a viral RNA encoding one or more viruses (e.g., one ormore strains of virus) and the second region comprises nucleotidesequence complementary to a viral RNA encoding one or more interferonagonist proteins. In one embodiment, the first region can comprisenucleotide sequence complementary to shared or conserved RNA sequencesof differing viral strains or classes or viral strains. Non-limitingexamples of viruses include Hepatitis C Virus (HCV), Hepatitis B Virus(HBV), Human Immunodeficiency Virus type 1 (HIV-1), HumanImmunodeficiency Virus type 2 (HIV-2), West Nile Virus (WNV),cytomegalovirus (CMV), respiratory syncytial virus (RSV), influenzavirus, rhinovirus, papillomavirus (HPV), Herpes Simplex Virus (HSV),severe acute respiratory virus (SARS), and other viruses such as HTLV.Non-limiting example of interferon agonist proteins include any proteinthat is capable of inhibition or suppressing RNA silencing (e.g., RNAbinding proteins such as E3L or NS1 or equivalents thereof, see forexample Li et al., 2004, PNAS, 101, 1350-1355)

In one embodiment, a multifunctional short interfering nucleic acid(multifunctional siNA) of the invention comprises a first region and asecond region, wherein the first region comprises nucleotide sequencecomplementary to a viral RNA and the second region comprises nucleotidesequence complementary to a cellular RNA that is involved in viralinfection and/or replication. Non-limiting examples of viruses includeHepatitis C Virus (HCV), Hepatitis B Virus (HBV), Human ImmunodeficiencyVirus type 1 (HIV-1), Human Immunodeficiency Virus type 2 (HIV-2), WestNile Virus (WNV), cytomegalovirus (CMV), respiratory syncytial virus(RSV), influenza virus, rhinovirus, papillomavirus (HPV), Herpes SimplexVirus (HSV), severe acute respiratory virus (SARS), and other virusessuch as HTLV. Non-limiting examples of cellular RNAs involved in viralinfection and/or replication include cellular receptors, cell surfacemolecules, cellular enzymes, cellular transcription factors, and/orcytokines, second messengers, and cellular accessory moleculesincluding, but not limited to, interferon agonsit proteins (e.g., E3L orNS1 or equivalents thereof, see for example Li et al., 2004, PNAS, 101,1350-1355), interferon regulatory factors (IRFs); cellular PKR proteinkinase (PKR); human eukaryotic initiation factors 2B (e1F2B gamma and/ore1F2gamma); human DEAD Box protein (DDX3); and cellular proteins thatbind to the poly(U) tract of the HCV 3′-UTR, such as polypyrimidinetract-binding protein, CD4 receptors, CXCR4 (Fusin; LESTR; NPY3R); CCR5(CKR-5, CMKRB5); CCR3 (CC-CKR-3, CKR-3, CMKBR3); CCR2 (CCR2b, CMKBR2);CCR1 (CKR1, CMKBR1); CCR4 (CKR-4); CCR8 (ChemR1, TER1, CMKBR8); CCR9(D6); CXCR2 (IL-8RB); STRL33 (Bonzo; TYMSTR); US28; V28 (CMKBRL1,CX3CR1, GPR13); GPR1; GPR15 (BOB); Apj (AGTRL1); ChemR23 receptors,Heparan Sulfate Proteoglycans, HSPG2; SDC2; SDC4; GPC1; SDC3; SDC1;Galactoceramides; Erythrocyte-expressed Glycolipids;N-myristoyltransferase (NMT, NMT2); Glycosylation Enzymes; gp-160Processing Enzymes (PCSK5); Ribonucleotide Reductase; PolyamineBiosynthesis enzymes; SP-1; NF-kappa B (NFKB2, RELA, and NFKB1); TumorNecrosis Factor-alpha (TNF-alpha); Interleukin 1 alpha (IL-1 alpha);Interleukin 6 (IL-6); Phospholipase C (PLC); Protein Kinase C (PKC),Cyclophilins, (PPID, PPIA, PPIE, PPIB, PPIF, PPIG, and PPIC); MitogenActivated Protein Kinase (MAP-Kinase, MAPK1); and ExtracellularSignal-Regulated Kinase (ERK-Kinase), (see for example Schang, 2002,Journal of Antimicrobial Chemotherapy, 50, 779-792 and Ludwig et al.,2003, Trends. Mol. Med., 9, 46-52).

In one embodiment, a double stranded multifunctional siNA molecule ofthe invention comprises a structure having Formula I(a):

5′-p-X Z X′-3′ 3′-Y′ Z Y-p-5′wherein each 5′-p-XZX′-3′ and 5′-p-YZY′-3′ are independently anoligonucleotide of length between about 20 nucleotides and about 300nucleotides, preferably between about 20 and about 200 nucleotides,about 20 and about 100 nucleotides, about 20 and about 40 nucleotides,about 20 and about 40 nucleotides, about 24 and about 38 nucleotides, orabout 26 and about 38 nucleotides; XZ comprises a nucleic acid sequencethat is complementary to a first target nucleic acid sequence; YZ is anoligonucleotide comprising nucleic acid sequence that is complementaryto a second target nucleic acid sequence; Z comprises nucleotidesequence of length about 1 to about 24 nucleotides (e.g. about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 nucleotides) that is complementary between regions XZ and YZ;X comprises nucleotide sequence of length about 1 to about 100nucleotides, preferably about 1 to about 21 nucleotides (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or21 nucleotides) that is complementary to nucleotide sequence present inregion Y′; Y comprises nucleotide sequence of length about 1 to about100 nucleotides, preferably about 1-about 21 nucleotides (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21nucleotides) that is complementary to nucleotide sequence present inregion X′; p comprises a terminal phosphate group that can independentlybe present or absent; each XZ and YZ independently is of lengthsufficient to stably interact (i.e., base pair) with the first andsecond target nucleic acid sequence, respectively, or a portion thereof.For example, each sequence X and Y can independently comprise sequencefrom about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14,15, 16, 17, 18, 19, 20, 21, or more) nucleotides in length that iscomplementary to a target nucleotide sequence in different targetnucleic acid molecules, such as target RNAs or a portion thereof. Inanother non-limiting example, the length of the nucleotide sequence of Xand Z together that is complementary to the first target nucleic acidsequence (e.g., RNA) or a portion thereof is from about 12 to about 21or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,or more). In another non-limiting example, the length of the nucleotidesequence of Y and Z together, that is complementary to the second targetnucleic acid sequence (e.g., RNA) or a portion thereof is from about 12to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18,19, 20, 21, or more). In one embodiment, the first target nucleic acidsequence and the second target nucleic acid sequence are present in thesame target nucleic acid molecule. In another embodiment, the firsttarget nucleic acid sequence and the second target nucleic acid sequenceare present in different target nucleic acid molecules. In oneembodiment, Z comprises a palindrome or a repeat sequence. In oneembodiment, the lengths of oligonucleotides X and X′ are identical. Inanother embodiment, the lengths of oligonucleotides X and X′ are notidentical. In one embodiment, the lengths of oligonucleotides Y and Y′are identical. In another embodiment, the lengths of oligonucleotides Yand Y′ are not identical. In one embodiment, the double strandedoligonucleotide construct of Formula I(a) includes one or more,specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches donot significantly diminish the ability of the double strandedoligonucleotide to inhibit target gene expression.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises structure having Formula II(a):

5′-p-X X′-3′ 3′-Y′ Y-p-5′wherein each 5′-p-XX′-3′ and 5′-p-YY′-3′ are independently anoligonucleotide of length between about 20 nucleotides and about 300nucleotides, preferably between about 20 and about 200 nucleotides,about 20 and about 100 nucleotides, about 20 and about 40 nucleotides,about 20 and about 40 nucleotides, about 24 and about 38 nucleotides, orabout 26 and about 38 nucleotides; X comprises a nucleic acid sequencethat is complementary to a first target nucleic acid sequence; Y is anoligonucleotide comprising nucleic acid sequence that is complementaryto a second target nucleic acid sequence; X comprises nucleotidesequence of length about 1 to about 100 nucleotides, preferably about 1to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) that iscomplementary to nucleotide sequence present in region Y′; Y comprisesnucleotide sequence of length about 1 to about 100 nucleotides,preferably about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21nucleotides) that is complementary to nucleotide sequence present inregion X′; p comprises a terminal phosphate group that can independentlybe present or absent; each X and Y independently is of length sufficientto stably interact (i.e., base pair) with the first and second targetnucleic acid sequence, respectively, or a portion thereof. For example,each sequence X and Y can independently comprise sequence from about 12to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18,19, 20, 21, or more) nucleotides in length that is complementary to atarget nucleotide sequence in different target nucleic acid molecules,such as target RNAs or a portion thereof. In one embodiment, the firsttarget nucleic acid sequence and the second target nucleic acid sequenceare present in the same target nucleic acid molecule. In anotherembodiment, the first target nucleic acid sequence and the second targetnucleic acid sequence are present in different target nucleic acidmolecules. In one embodiment, Z comprises a palindrome or a repeatsequence. In one embodiment, the lengths of oligonucleotides X and X′are identical. In another embodiment, the lengths of oligonucleotides Xand X′ are not identical. In one embodiment, the lengths ofoligonucleotides Y and Y′ are identical. In another embodiment, thelengths of oligonucleotides Y and Y′ are not identical. In oneembodiment, the double stranded oligonucleotide construct of FormulaI(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to theextent such mismatches do not significantly diminish the ability of thedouble stranded oligonucleotide to inhibit target gene expression.

In one embodiment, regions X and Y of multifunctional siNA molecule ofthe invention (e.g., having any of Formula I or II), are complementaryto different target nucleic acid sequences that are portions of the sametarget nucleic acid molecule. In one embodiment, such as target nucleicacid sequences are at different locations within the coding region of aRNA transcript. In one embodiment, such target nucleic acid sequencescomprise coding and non-coding regions of the same RNA transcript. Inone embodiment, such target nucleic acid sequences comprise regions ofalternately spliced transcripts or precursors of such alternatelyspliced transcripts.

In one embodiment, a multifunctional siNA molecule having any of FormulaI or II can comprise chemical modifications as described herein withoutlimitation, such as, for example, nucleotides having any of FormulaeIII-IX described herein, stabilization chemistries as described in TableVIII, or any other combination of modified nucleotides andnon-nucleotides as described in the various embodiments herein.

In one embodiment, the palidrome or repeat sequence or modifiednucleotide (e.g. nucleotide with a modified base, such as 2-amino purineor a universal base) in Z of multifunctional siNA constructs havingFormula I(a) or I(b), comprises chemically modified nucleotides that areable to interact with a portion of the target nucleic acid sequence(e.g., modified base analogs that can form Watson Crick base pairs ornon-Watson Crick base pairs).

In one embodiment, a multifunctional siNA molecule of the invention, forexample each strand of a multifunctional siNA having Formula I or II,independently comprises about 15 to about 40 nucleotides (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, amultifunctional siNA molecule of the invention comprises one or morechemical modifications. In a non-limiting example, the introduction ofchemically modified nucleotides and/or non-nucleotides into nucleic acidmolecules of the invention provides a powerful tool in overcomingpotential limitations of in vivo stability and bioavailability inherentto unmodified RNA molecules that are delivered exogenously. For example,the use of chemically modified nucleic acid molecules can enable a lowerdose of a particular nucleic acid molecule for a given therapeuticeffect since chemically modified nucleic acid molecules tend to have alonger half-life in serum or in cells or tissues. Furthermore, certainchemical modifications can improve the bioavailability and/or potency ofnucleic acid molecules by not only enhancing half-life but alsofacilitating the targeting of nucleic acid molecules to particularorgans, cells or tissues and/or improving cellular uptake of the nucleicacid molecules. Therefore, even if the activity of a chemically modifiednucleic acid molecule is reduced in vitro as compared to anative/unmodified nucleic acid molecule, for example when compared to anunmodified RNA molecule, the overall activity of the modified nucleicacid molecule can be greater than the native or unmodified nucleic acidmolecule due to improved stability, potency, duration of effect,bioavailability and/or delivery of the molecule.

In one embodiment, the invention features chemically modifiedmultifunctional siNA constructs having specificity for more than onetarget nucleic acid molecules, such as in an in vitro system, cell ororganism. Non-limiting examples of such chemical modificationsindependently include without limitation phosphate backbone modification(e.g. phosphorothioate internucleotide linkages), nucleotide sugarmodification (e.g., 2′-O-methyl nucleotides, 2′-O-allyl nucleotides,2′-deoxy-2′-fluoro nucleotides, 2′-deoxyribonucleotides), nucleotidebase modification (e.g., “universal base” containing nucleotides,5-C-methyl nucleotides), and non-nucleotide modification (e.g., abasicnucleotides, inverted deoxyabasic residue) or a combination of thesemodifications. These and other chemical modifications, when used invarious multifunctional siNA constructs, can preserve biologicalactivity of the multifunctional siNAs in vivo while at the same time,dramatically increasing the serum stability, potency, duration of effectand/or specificity of these compounds.

In one embodiment, a multifunctional siNA molecule of the invention cangenerally comprise modified nucleotides from about 5 to about 100% ofthe nucleotide positions (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of thenucleotide positions may be modified). The actual percentage of modifiednucleotides present in a given multifunctional siNA molecule depends onthe total number of nucleotides present in the multifunctional siNA. Ifthe multifunctional siNA molecule is single stranded, the percentmodification can be based upon the total number of nucleotides presentin the single stranded multifunctional siNA molecules. Likewise, if themultifunctional siNA molecule is double stranded, the percentmodification can be based upon the total number of nucleotides presentin both strands. In addition, the actual percentage of modifiednucleotides present in a given multifunctional siNA molecule can alsodepend on the total number of purine and pyrimidine nucleotides presentin the multifunctional siNA, for example, wherein all pyrimidinenucleotides and/or all purine nucleotides present in the multifunctionalsiNA molecule are modified.

In one embodiment, a multifunctional siNA duplex molecule can comprisemismatches (e.g., 1, 2, 3, 4 or 5 mismatches), bulges, loops, or wobblebase pairs, for example, to modulate or regulate the ability of themultifunctional siNA molecule to mediate inhibition of gene expression.Mismatches, bulges, loops, or wobble base pairs can be introduced intothe multifunctional siNA duplex molecules to the extent such mismatches,bulges, loops, or wobble base pairs do not significantly impair theability of the multifunctional siNAs to mediate inhibition of targetgene expression. Such mismatches, bulges, loops, or wobble base pairscan be present in regions of the multifunctional siNA duplex that do notsignificantly impair the ability of such multifunctional siNAs tomediate inhibition of gene expression, for example, mismatches can bepresent at the terminal regions of the duplex or at one or positions inthe internal regions of the duplex. Similarly, the wobble base pairscan, for example, be at the terminal base paired region(s) of the duplexor in the internal regions or in the regions where self complementary,palindromic, or repeat sequences are present within the multifunctionalsiNA.

In one embodiment, a multifunctional siNA molecule of the invention cancomprise one or more (e.g., about 1, 2, 3, 4, or 5) phosphorothioateinternucleotide linkages at the 3′-end of the multifunctional siNAmolecule.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a 3′ nucleotide overhang region, which includes one or more(e.g., about 1, 2, 3, 4) unpaired nucleotides when the multifunctionalsiNA is in duplex form. In a non-limiting example, the multifunctionalsiNA duplex with overhangs includes a fewer number of base pairs thanthe number of nucleotides present in each strand of the multifunctionalsiNA molecule (e.g., a multifunctional siNA 18 nucleotides in lengthforming a 16 base-paired duplex with 2 nucleotide overhangs at the 3′ends). Such blunt-end multifunctional siNA duplex may optionally includeone or more mismatches, wobble base-pairs or nucleotide bulges. The3′-terminal nucleotide overhangs of a multifunctional siNA molecule ofthe invention can comprise ribonucleotides or deoxyribonucleotides thatare chemically-modified at a nucleic acid sugar, base, or phosphatebackbone. The 3′-terminal nucleotide overhangs can comprise one or moreuniversal base nucleotides. The 3′-terminal nucleotide overhangs cancomprise one or more acyclic nucleotides or non-nucleotides.

In one embodiment, a multifunctional siNA molecule of the invention induplex form comprises blunt ends, i.e., the ends do not include anyoverhanging nucleotides. For example, a multifunctional siNA duplexmolecule of the invention comprising modifications described herein(e.g., comprising modifications having Formulae III-IX ormultifunctional siNA constructs comprising Stab1-Stab22 or anycombination thereof) and/or any length described herein, has blunt endsor ends with no overhanging nucleotides.

In one embodiment, any multifunctional siNA duplex of the invention cancomprise one or more blunt ends, i.e. where a blunt end does not haveany overhanging nucleotides. In a non-limiting example, a blunt endedmultifunctional siNA duplex includes the same number of base pairs asthe number of nucleotides present in each strand of the multifunctionalsiNA molecule (e.g., a multifunctional siNA 18 nucleotides in lengthforming an 18 base-paired duplex). Such blunt-end multifunctional siNAduplex may optionally include one or more mismatches, wobble base-pairsor nucleotide bulges.

By “blunt ends” is meant symmetric termini or termini of amultifunctional siNA duplex having no overhanging nucleotides. The twostrands of a multifunctional siNA duplex molecule align with each otherwithout over-hanging nucleotides at the termini. For example, a bluntended multifunctional siNA duplex comprises terminal nucleotides thatare complementary between the two strands of the multifunctional siNAduplex.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises no ribonucleotides and is capable of down-regulatingexpression of more than one target gene in vitro or in vivo.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises sequence wherein one or more pyrimidine nucleotides present inthe multifunctional siNA sequence is a 2′-deoxy-2′-fluoro pyrimidinenucleotide. In another embodiment, a multifunctional siNA molecule ofthe invention comprises sequence wherein all pyrimidine nucleotidespresent in the multifunctional siNA sequence are 2′-deoxy-2′-fluoropyrimidine nucleotides. Such multifunctional siNA sequences can furthercomprise differing nucleotides or non-nucleotide caps described herein,such as deoxynucleotides, inverted nucleotides, abasic moieties,inverted abasic moieties, and/or any other modification shown in FIG. 10or those modifications generally known in the art that can be introducedinto nucleic acid molecules, to the extent any modification to themultifunctional siNA molecule does not significantly impair the abilityof the multifunctional siNA molecule to mediate inhibition of geneexpression.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises sequence wherein one or more purine nucleotides present in themultifunctional siNA sequence is a 2′-sugar modified purine, (e.g.,2′-O-methyl purine nucleotide, 2′-O-allyl purine nucleotide, or2′-methoxy-ethoxy purine nucleotides). In another embodiment, amultifunctional siNA molecule of the invention comprises sequencewherein all purine nucleotides present in the multifunctional siNAsequence are 2′-sugar modified purines, (e.g., 2′-O-methyl purinenucleotides, 2′-O-allyl purine nucleotides, or 2′-methoxy-ethoxy purinenucleotides).

In one embodiment, a multifunctional siNA molecule of the inventioncomprises sequence wherein one or more purine nucleotides present in themultifunctional siNA sequence is a 2′-deoxy purine nucleotide. Inanother embodiment, a multifunctional siNA molecule of the inventioncomprises sequence wherein all purine nucleotides present in themultifunctional siNA sequence are 2′-deoxy purine nucleotides.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises sequence wherein one or more purine nucleotides present in themultifunctional siNA sequence is a 2′-deoxy-2′-fluoro purine nucleotide.In another embodiment, a multifunctional siNA molecule of the inventioncomprises sequence wherein all purine nucleotides present in themultifunctional siNA sequence are 2′-deoxy-2′-fluoro purine nucleotides.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises sequence wherein the multifunctional siNA sequence includes aterminal cap moiety at the 3′-end of one or both of the multifunctionalsiNA sequences. In another embodiment, the terminal cap moiety is aninverted deoxy abasic moiety or any other modification shown in FIG. 9or those modifications generally known in the art that can be introducedinto nucleic acid molecules, to the extent any modification to themultifunctional siNA molecule does not significantly impair the abilityof the multifunctional siNA molecule to mediate inhibition of geneexpression.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises sequence wherein the multifunctional siNA sequence includes aterminal cap moiety at the 3′ end of the multifunctional siNA sequence.In another embodiment, the terminal cap moiety is an inverted deoxyabasic moiety or any other modification shown in FIG. 9 or thosemodifications generally known in the art that can be introduced intonucleic acid molecules, to the extent any modification to themultifunctional siNA molecule does not significantly impair the abilityof the multifunctional siNA molecule to mediate inhibition of geneexpression.

In one embodiment, a multifunctional siNA molecule of the invention hasactivity that modulates expression of RNA encoded by more than one gene.Because many genes can share some degree of sequence homology with eachother, multifunctional siNA molecules can be designed to target a classof genes (and associated receptor or ligand genes) or alternatelyspecific genes by selecting sequences that are either shared amongstdifferent gene targets or alternatively that are unique for a specificgene target. Therefore, in one embodiment, each complementary region ofa multifunctional siNA molecule of the invention can be designed totarget conserved regions of a RNA sequence having homology betweenseveral genes or genomes (e.g. viral genome, such as HIV, HCV, HBV, SARSand others) so as to target several genes or gene families (e.g.,different gene isoforms, splice variants, mutant genes etc.) with onemultifunctional siNA molecule. In another embodiment, each complementaryregion of a multifunctional siNA molecule of the invention can bedesigned to target a sequence that is unique to a specific RNA sequenceof a specific gene or genome (e.g. viral genome, such as HIV, HCV, HBV,SARS and others). The expression of any target nucleic acid having knownsequence can be modulated by multifunctional siNA molecules of theinvention (see for example McSwiggen et al., WO 03/74654 incorporated byreference herein in its entirety for a list of mammalian and viraltargets).

In one embodiment, a multifunctional siNA molecule of the invention doesnot contain any ribonucleotides. In another embodiment, amultifunctional siNA molecule of the invention comprises one or moreribonucleotides.

In one embodiment, the multifunctional siNA molecule of the inventiondoes not include any chemical modification. In another embodiment, themultifunctional siNA molecule of the invention is RNA comprising nochemical modifications. In another embodiment, the multifunctional siNAmolecule of the invention is RNA comprising two deoxyribonucleotides atthe 3′-end. In another embodiment, the multifunctional siNA molecule ofthe invention is RNA comprising a 3′-cap structure (e.g., inverteddeoxynucleotide, inverted deoxy abasic moiety, a thymidine dinucleotideresidues or a thymidine dinucleotide with a phosphorothioateinternucleotide linkage, and the like).

In one embodiment of the present invention, each sequence of amultifunctional siNA molecule is independently about 18 to about 300nucleotides in length, in specific embodiments about 18-200 nucleotidesin length, preferably 18-150 nucleotides in length, more specifically18-100 nucleotides in length. In another embodiment, the multifunctionalsiNA duplexes of the invention independently comprise about 18 to about300 base pairs (e.g., about 18-200, 18-150, 18-100, 18-75, 18-50, 18-34or 18-30 base pairs).

In one embodiment, the invention features a multifunctional siNAmolecule that inhibits the replication of a virus (e.g., as plant virussuch as tobacco mosaic virus, or mammalian virus, such as hepatitis Cvirus, human immunodeficiency virus, hepatitis B virus, herpes simplexvirus, cytomegalovirus, human papilloma virus, rhino virus, respiratorysyncytial virus, SARS, or influenza virus).

In one embodiment, the invention features a medicament comprising amultifunctional siNA molecule of the invention.

In one embodiment, the invention features an active ingredientcomprising a multifunctional siNA molecule of the invention.

In one embodiment, the invention features the use of a multifunctionalsiNA molecule of the invention to down-regulate expression of a targetgene.

In one embodiment, the invention features a composition comprising amultifunctional siNA molecule of the invention and a pharmaceuticallyacceptable carrier or diluent.

In one embodiment, the invention features a method of increasing thestability of a multifunctional siNA molecule against cleavage byribonucleases or other nucleases, comprising introducing at least onemodified nucleotide into the multifunctional siNA molecule, wherein themodified nucleotide is for example a 2′-deoxy-2′-fluoro nucleotide. Inanother embodiment, all pyrimidine nucleotides present in themultifunctional siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. Inanother embodiment, the modified nucleotides in the multifunctional siNAinclude at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluorouridine nucleotide. In another embodiment, the modified nucleotides inthe multifunctional siNA include at least one 2′-fluoro cytidine and atleast one 2′-deoxy-2′-fluoro uridine nucleotides. In another embodiment,all uridine nucleotides present in the multifunctional siNA are2′-deoxy-2′-fluoro uridine nucleotides. In another embodiment, allcytidine nucleotides present in the multifunctional siNA are2′-deoxy-2′-fluoro cytidine nucleotides. In another embodiment, alladenosine nucleotides present in the multifunctional siNA are2′-deoxy-2′-fluoro adenosine nucleotides. In another embodiment, allguanosine nucleotides present in the multifunctional siNA are2′-deoxy-2′-fluoro guanosine nucleotides. The multifunctional siNA canfurther comprise at least one modified internucleotidic linkage, such asphosphorothioate linkage or phosphorodithioate linkage. In anotherembodiment, the 2′-deoxy-2′-fluoronucleotides are present atspecifically selected locations in the multifunctional siNA that aresensitive to cleavage by ribonucleases or other nucleases, such aslocations having pyrimidine nucleotides or terminal nucleotides. Themultifunctional siNA molecules of the invention can be modified toimprove stability, pharmacokinetic properties, in vitro or in vivodelivery, localization and/or potency by methods generally known in theart (see for example Beigelman et al., WO WO 03/70918 incorporated byreference herein in its entirety including the drawings).

In one embodiment, a multifunctional siNA molecule of the inventioncomprises nucleotide sequence having complementarity to nucleotidesequence of RNA or a portion thereof encoded by the target nucleic acidor a portion thereof.

In one embodiment, the invention features a multifunctional siNAmolecule having a first region and a second region, wherein the secondregion comprises nucleotide sequence that is an inverted repeat sequenceof the nucleotide sequence of the first region, wherein the first regionis complementary to nucleotide sequence of a target nucleic acid (e.g.,RNA) or a portion thereof (see for example FIGS. 1 and 2 for anillustration of non-limiting examples of multifunctional siNA moleculesof the instant invention).

One embodiment of the invention provides an expression vector comprisinga nucleic acid sequence encoding at least one multifunctional siNAmolecule of the invention in a manner that allows expression of themultifunctional siNA sequence. Another embodiment of the inventionprovides a mammalian cell comprising such an expression vector. Themammalian cell can be a human cell.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) nucleotides comprising a backbone modified internucleotide linkagehaving Formula III:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally-occurring orchemically-modified, each X and Y is independently O, S, N, alkyl, orsubstituted alkyl, each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W,X, Y, and Z are optionally not all O. In another embodiment, a backbonemodification of the invention comprises a phosphonoacetate and/orthiophosphonoacetate internucleotide linkage (see for example Sheehan etal., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula III, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present anywhere in the multifunctional siNA sequence.Non-limiting examples of such phosphate backbone modifications arephosphorothioate and phosphorodithioate. The multifunctional siNAmolecules of the invention can comprise one or more (e.g., about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modified internucleotidelinkages having Formula III at the 3′-end, the 5′-end, or both of the 3′and 5′-ends of the multifunctional siNA sequence. In anothernon-limiting example, an exemplary multifunctional siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically-modifiedinternucleotide linkages having Formula III. In yet another non-limitingexample, an exemplary multifunctional siNA molecule of the invention cancomprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) purine nucleotides with chemically-modified internucleotidelinkages having Formula III. In another embodiment, a multifunctionalsiNA molecule of the invention having internucleotide linkage(s) ofFormula III also comprises a chemically-modified nucleotide ornon-nucleotide having any of Formulae III-IX.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) nucleotides or non-nucleotides having Formula IV:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula III or IV;R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, 2-aminopurine,2-amino-1,6-dihydropurine or any other non-naturally occurring base thatcan be complementary or non-complementary to target RNA or anon-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula IV canbe present anywhere in the multifunctional siNA sequence. Themultifunctional siNA molecules of the invention can comprise one or morechemically-modified nucleotide or non-nucleotide of Formula IV at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the multifunctionalsiNA sequence. For example, an exemplary multifunctional siNA moleculeof the invention can comprise about 1 to about 5 or more (e.g., about 1,2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotidesof Formula IV at the 5′-end of the multifunctional siNA sequence. Inanother non-limiting example, an exemplary multifunctional siNA moleculeof the invention can comprise about 1 to about 5 or more (e.g., about 1,2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotidesof Formula IV at the 3′-end of the multifunctional siNA sequence.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) nucleotides or non-nucleotides having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula III or IV;R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be employed to be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula V can bepresent anywhere in the multifunctional siNA sequence. Themultifunctional siNA molecules of the invention can comprise one or morechemically-modified nucleotide or non-nucleotide of Formula V at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the multifunctionalsiNA sequence. For example, an exemplary multifunctional siNA moleculeof the invention can comprise about 1 to about 5 or more (e.g., about 1,2, 3, 4, 5, or more) chemically-modified nucleotide(s) ornon-nucleotide(s) of Formula V at the 5′-end of multifunctional siNAsequence. In anther non-limiting example, an exemplary multifunctionalsiNA molecule of the invention can comprise about 1 to about 5 or more(e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide ornon-nucleotide of Formula V at the 3′-end of the multifunctional siNAsequence.

In another embodiment, a multifunctional siNA molecule of the inventioncomprises a nucleotide having Formula IV or V, wherein the nucleotidehaving Formula IV or V is in an inverted configuration. For example, thenucleotide having Formula IV or V is connected to the multifunctionalsiNA construct in a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such asat the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or bothmultifunctional siNA strands.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a 5′-terminal phosphate group having Formula VI:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo; wherein each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or alkylhalo oracetyl; and/or wherein W, X, Y and Z are optionally not all O.

In another embodiment, a multifunctional siNA molecule of the inventioncomprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) canbe anywhere in the multifunctional siNA sequence. In addition, the 2′-5′internucleotide linkage(s) can be present at various other positionswithin the multifunctional siNA sequence, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of apyrimidine nucleotide in the multifunctional siNA molecule can comprisea 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a purine nucleotidein the multifunctional siNA molecule can comprise a 2′-5′internucleotide linkage.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) abasic moiety, for example a compound having Formula VII:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula III or IV; R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) inverted nucleotide or abasic moiety, for example a compoundhaving Formula VIII:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula III or IV; R9 is O, S, CH2, S═O, CHF, or CF2, and either R3, R5,R8 or R13 serve as points of attachment to the multifunctional siNAmolecule of the invention.

In another embodiment, a multifunctional siNA molecule of the inventioncomprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) substituted polyalkyl moieties, for example a compound havingFormula IX:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or a group havingFormula III, and R1, R2 or R3 serves as points of attachment to themultifunctional siNA molecule of the invention.

In another embodiment, the invention features a compound having FormulaIX, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises 0and is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a multifunctional siNA moleculeof the invention. This modification is referred to herein as “glyceryl”(for example modification 6 in FIG. 9).

In another embodiment, a moiety having any of Formula VII, VIII or IX ofthe invention is at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of a multifunctional siNA molecule of the invention. In anotherembodiment, a moiety having any of Formula VII, VIII or IX of theinvention is at the 3′-end of a multifunctional siNA molecule of theinvention.

In another embodiment, a multifunctional siNA molecule of the inventioncomprises an abasic residue having Formula VII or VIII, wherein theabasic residue having Formula VII or VIII is connected to themultifunctional siNA construct in a 3-3′, 3-2′, 2-3′, or 5-5′configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the multifunctional siNA molecule. In another embodiment, amultifunctional siNA molecule of the invention comprises an abasicresidue having Formula VII or VIII, wherein the abasic residue havingFormula VII or VIII is connected to the multifunctional siNA constructin a 3-3′ or 3-2′ configuration at the 3′-end of the multifunctionalsiNA molecule.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) locked nucleic acid (LNA) nucleotides, for example at the 5′-end,the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, ofthe multifunctional siNA molecule.

In another embodiment, a multifunctional siNA molecule of the inventioncomprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) acyclic nucleotides, for example at the 5′-end, the 3′-end, bothof the 5′ and 3′-ends, or any combination thereof, of themultifunctional siNA molecule. In another embodiment, a multifunctionalsiNA molecule of the invention comprises one or more (e.g., about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides at the 3′-end ofthe multifunctional siNA molecule.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a terminal cap moiety, (see for example FIG. 9) such as aninverted deoxyabasic moiety or inverted nucleotide, at the 3′-end of oneor both strands of the multifunctional siNA molecule.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises sequence wherein any (e.g., one or more or all) pyrimidinenucleotides present in the multifunctional siNA are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and where any (e.g., one or more or all) purine nucleotides present inthe multifunctional siNA are 2′-deoxy purine nucleotides (e.g., whereinall purine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides). Themultifunctional siNA can further comprise terminal cap modifications asdescribed herein.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises sequence wherein any (e.g., one or more or all) pyrimidinenucleotides present in the multifunctional siNA are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and where any (e.g., one or more or all) purine nucleotides present inthe multifunctional siNA are 2′-O-methyl purine nucleotides (e.g.,wherein all purine nucleotides are 2′-O-methyl purine nucleotides oralternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides). The multifunctional siNA can further comprise terminal capmodifications as described herein.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises sequence wherein any (e.g., one or more or all) pyrimidinenucleotides present in the multifunctional siNA are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe multifunctional siNA are selected from the group consisting of2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methylnucleotides (e.g., wherein all purine nucleotides are selected from thegroup consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA)nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and2′-O-methyl nucleotides or alternately a plurality of purine nucleotidesare selected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides).

In another embodiment, a multifunctional siNA molecule of the inventioncomprises modified nucleotides having properties or characteristicssimilar to naturally occurring ribonucleotides. For example, theinvention features multifunctional siNA molecules including modifiednucleotides having a Northern conformation (e.g., Northernpseudorotation cycle, see for example Saenger, Principles of NucleicAcid Structure, Springer-Verlag ed., 1984). As such, chemically modifiednucleotides present in the multifunctional siNA molecules of theinvention are resistant to nuclease degradation while at the same timemaintaining the capacity to modulate gene expression. Non-limitingexamples of nucleotides having a northern configuration include lockednucleic acid (LNA) nucleotides (e.g.,2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy(MOE) nucleotides; 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides,2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, and 2′-O-methylnucleotides.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a conjugate attached to the multifunctional siNA molecule. Forexample, the conjugate can be attached to the multifunctional siNAmolecule via a covalent attachment. In one embodiment, the conjugate isattached to the multifunctional siNA molecule via a biodegradablelinker. In one embodiment, the conjugate molecule is attached at the3′-end of the multifunctional siNA molecule. In another embodiment, theconjugate molecule is attached at the 5′-end of the multifunctional siNAmolecule. In yet another embodiment, the conjugate molecule is attachedat both the 3′-end and 5′-end of the multifunctional siNA molecule, orany combination thereof. In one embodiment, the conjugate molecule ofthe invention comprises a molecule that facilitates delivery of amultifunctional siNA molecule into a biological system, such as a cell.In another embodiment, the conjugate molecule attached to thechemically-modified multifunctional siNA molecule is a polyethyleneglycol, human serum albumin, or a ligand for a cellular receptor thatcan mediate cellular uptake. Examples of specific conjugate moleculescontemplated by the instant invention that can be attached tomultifunctional siNA molecules are described in Vargeese et al., U.S.Ser. No. 10/201,394, incorporated by reference herein. The type ofconjugates used and the extent of conjugation of multifunctional siNAmolecules of the invention can be evaluated for improved pharmacokineticprofiles, bioavailability, and/or stability of multifunctional siNAconstructs while at the same time maintaining the ability of themultifunctional siNA to modulate gene expression. As such, one skilledin the art can screen multifunctional siNA constructs that are modifiedwith various conjugates to determine whether the multifunctional siNAconjugate complex possesses improved properties while maintaining theability to modulate gene expression, for example in animal models as aregenerally known in the art.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a non-nucleotide linker, such as an abasic nucleotide,polyether, polyamine, polyamide, peptide, carbohydrate, lipid,polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycolssuch as those having between 2 and 100 ethylene glycol units). Specificexamples include those described by Seela and Kaiser, Nucleic Acids Res.1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz,J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem.Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 andBiochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990,18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschkeet al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991,30:9914; Arnold et al., International Publication No. WO 89/02439; Usmanet al., International Publication No. WO 95/06731; Dudycz et al.,International Publication No. WO 95/11910 and Ferentz and Verdine, J.Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by referenceherein. A “non-nucleotide” further means any group or compound that canbe incorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound can be abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine, for example at the C1 position of the sugar.

In one embodiment, the invention features a multifunctional siNAmolecule that does not require the presence of a 2′-OH group(ribonucleotide) to be present within the multifunctional siNA moleculeto support inhibition or modulation of gene expression of target nucleicacids.

In one embodiment, the invention features a method for modulating theexpression of one or more genes within a cell comprising: (a)synthesizing a multifunctional siNA molecule of the invention, which canbe chemically-modified or unmodified, wherein the multifunctional siNAcomprises sequences complementary to one or more RNAs of the gene(s) orportions thereof; and (b) introducing the multifunctional siNA moleculeinto a cell under conditions suitable to modulate the expression of thegene(s) in the cell.

In one embodiment, the invention features a method for modulating theexpression of a gene within a cell comprising: (a) synthesizing amultifunctional siNA molecule of the invention, which can bechemically-modified or unmodified, wherein the multifunctional siNAcomprises a first strand and a second strand that are complementary toeach other, and wherein the first strand comprises a region havingsequence complementarity to a first portion of a RNA of the gene or aportion thereof and the second strand comprises a region having sequencecomplementarity to a second portion of the RNA of the gene or a portionthereof; and (b) introducing the multifunctional siNA molecule into acell under conditions suitable to modulate the expression of the gene inthe cell. The first and second portions of the RNA can comprise, forexample, coding and/or non-coding sequences of the gene.

In one embodiment, the invention features a method for modulating theexpression of a gene within a cell comprising: (a) synthesizing amultifunctional siNA molecule of the invention, which can bechemically-modified or unmodified, wherein the multifunctional siNAcomprises a first strand and a second strand that are complementary toeach other, and wherein the first strand comprises a region havingsequence complementarity to a first portion of a RNA of the gene or aportion thereof and the second strand comprises a region having sequencecomplementarity to a second RNA that regulates the expression of thegene or a portion thereof; and (b) introducing the multifunctional siNAmolecule into a cell under conditions suitable to modulate theexpression of the gene in the cell. The first RNA can comprise forexample a coding or non-coding sequence of the gene. The second RNA cancomprise for example an enhancer region, a tRNA, a RNA encoding anenhancer element, a RNA encoding a transcription factor, a micro RNA,stRNA, or other non-coding RNA that is involved in the expression of thetarget gene.

In one embodiment, the invention features a method for modulating theexpression of more than one gene within a cell comprising: (a)synthesizing a multifunctional siNA molecule of the invention, which canbe chemically-modified or unmodified, wherein the multifunctional siNAcomprises a first strand and a second strand that are complementary toeach other, and wherein the first strand comprises a region havingsequence complementarity to a RNA of a first gene or a portion thereofand the second strand comprises a region having sequence complementarityto a RNA of a second gene or a portion thereof; and (b) introducing themultifunctional siNA molecule into a cell under conditions suitable tomodulate the expression of the genes in the cell. The RNA of the firstand second genes can independently comprise coding and/or non-codingsequences of the genes. In one embodiment, the first gene encodes one ormore cytokines and the second gene encodes one or more receptors of thecytokine(s). In one embodiment, the first gene encodes one or morestrains of a virus and the second gene encodes one or strains of thesame virus. In one embodiment, the first gene encodes one or morestrains of a virus and the second gene encodes one or strains of adifferent virus. In one embodiment, the first gene encodes one or morestrains of a virus and the second gene encodes one or more cellularfactors involved in infection or replication of the virus. In oneembodiment, the first gene encodes a first protein and the second geneencodes a second protein that are involved in a common biologic pathway.In one embodiment, the first gene encodes a first protein and the secondgene encodes a second protein that are involved in divergent biologicpathways.

In one embodiment, multifunctional siNA molecules of the invention areused as reagents in ex vivo applications. For example, multifunctionalsiNA reagents are introduced into tissue or cells that are transplantedinto a subject for therapeutic effect. The cells and/or tissue can bederived from an organism or subject that later receives the explant, orcan be derived from another organism or subject prior totransplantation. The multifunctional siNA molecules can be used tomodulate the expression of one or more genes in the cells or tissue,such that the cells or tissue obtain a desired phenotype or are able toperform a function when transplanted in vivo. In one embodiment, certaintarget cells from a patient are extracted. These extracted cells arecontacted with multifunctional siNAs targeting a specific nucleotidesequence within the cells under conditions suitable for uptake of themultifunctional siNAs by these cells (e.g., using delivery reagents suchas cationic lipids, liposomes and the like or using techniques such aselectroporation to facilitate the delivery of multifunctional siNAs intocells). The cells are then reintroduced back into the same patient orother patients. Non-limiting examples of ex vivo applications includeuse in organ/tissue transplant, tissue grafting, or treatment ofpulmonary disease (e.g., restenosis) or prevent neointimal hyperplasiaand atherosclerosis in vein grafts. Such ex vivo applications may alsobe used to treat conditions associated with coronary and peripheralbypass graft failure, for example, such methods can be used inconjunction with peripheral vascular bypass graft surgery and coronaryartery bypass graft surgery. Additional applications include transplantsto treat CNS lesions or injury, including use in treatment ofneurodegenerative conditions such as Alzheimer's disease, Parkinson'sDisease, Epilepsy, Dementia, Huntington's disease, or amyotrophiclateral sclerosis (ALS).

In one embodiment, the invention features a method of modulating theexpression of one or more genes in a tissue explant comprising: (a)synthesizing a multifunctional siNA molecule of the invention, which canbe chemically-modified or unmodified, wherein the multifunctional siNAcomprises sequences complementary to one or more RNAs of the gene(s) orportions thereof; and (b) introducing the multifunctional siNA moleculeinto a cell of the tissue explant derived from a particular organismunder conditions suitable to modulate the expression of the gene(s) inthe tissue explant. In another embodiment, the method further comprisesintroducing the tissue explant back into the organism the tissue wasderived from or into another organism under conditions suitable tomodulate the expression of the gene(s) in that organism.

In one embodiment, the invention features a method of modulating theexpression of a gene in a tissue explant comprising: (a) synthesizing amultifunctional siNA molecule of the invention, which can bechemically-modified or unmodified, wherein the multifunctional siNAcomprises a first strand and a second strand that are complementary toeach other, and wherein the first strand comprises a region havingsequence complementarity to a first portion of a RNA of the gene or aportion thereof and the second strand comprises a region having sequencecomplementarity to a second portion of the RNA of the gene or a portionthereof; and (b) introducing the multifunctional siNA molecule into acell of the tissue explant derived from a particular organism underconditions suitable to modulate the expression of the gene in the tissueexplant. In another embodiment, the method further comprises introducingthe tissue explant back into the organism the tissue was derived from orinto another organism under conditions suitable to modulate theexpression of the gene in that organism. The first and second portionsof the RNA can comprise, for example, coding and/or non-coding sequencesof the gene.

In another embodiment, the invention features a method of modulating theexpression of more than one gene in a tissue explant comprising: (a)synthesizing a multifunctional siNA molecule of the invention, which canbe chemically-modified or unmodified, wherein the multifunctional siNAcomprises a first strand and a second strand that are complementary toeach other, and wherein the first strand comprises a region havingsequence complementarity to a RNA of a first gene or a portion thereofand the second strand comprises a region having sequence complementarityto a second gene or a portion thereof; and (b) introducing themultifunctional siNA molecule(s) into a cell of the tissue explantderived from a particular organism under conditions suitable to modulatethe expression of the genes in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of thegenes in that organism. The RNA of the first and second genes canindependently comprise coding and/or non-coding sequences of the genes.In one embodiment, the first gene encodes one or more cytokines and thesecond gene encodes one or more receptors of the cytokine(s). In oneembodiment, the first gene encodes one or more strains of a virus andthe second gene encodes one or strains of the same virus. In oneembodiment, the first gene encodes one or more strains of a virus andthe second gene encodes one or strains of a different virus. In oneembodiment, the first gene encodes one or more strains of a virus andthe second gene encodes one or more cellular factors involved ininfection or replication of the virus. In one embodiment, the first geneencodes a first protein and the second gene encodes a second proteinthat are involved in a common biologic pathway. In one embodiment, thefirst gene encodes a first protein and the second gene encodes a secondprotein that are involved in divergent biologic pathways.

In one embodiment, the invention features a method for modulating theexpression of one or more genes within an organism comprising: (a)synthesizing a multifunctional siNA molecule of the invention, which canbe chemically-modified or unmodified, wherein the multifunctional siNAcomprises sequences complementary to one or more RNAs of the gene(s) orportions thereof; and (b) introducing the multifunctional siNA moleculeinto the organism under conditions suitable to modulate the expressionof the gene(s) in the organism.

In one embodiment, the invention features a method for modulating theexpression of a gene within an organism comprising: (a) synthesizing amultifunctional siNA molecule of the invention, which can bechemically-modified or unmodified, wherein the multifunctional siNAcomprises a first strand and a second strand that are complementary toeach other, and wherein the first strand comprises a region havingsequence complementarity to a first portion of a RNA of the gene or aportion thereof and the second strand comprises a region having sequencecomplementarity to a second portion of the RNA of the gene or a portionthereof; and (b) introducing the multifunctional siNA molecule into theorganism under conditions suitable to modulate the expression of thegene in the cell. The first and second portions of the RNA can comprise,for example, coding and/or non-coding sequences of the gene.

In one embodiment, the invention features a method for modulating theexpression of a gene within an organism comprising: (a) synthesizing amultifunctional siNA molecule of the invention, which can bechemically-modified or unmodified, wherein the multifunctional siNAcomprises a first strand and a second strand that are complementary toeach other, and wherein the first strand comprises a region havingsequence complementarity to a first portion of a RNA of the gene or aportion thereof and the second strand comprises a region having sequencecomplementarity to a second RNA that regulates the expression of thegene or a portion thereof; and (b) introducing the multifunctional siNAmolecule into the organism under conditions suitable to modulate theexpression of the gene in the organism. The first RNA can comprise forexample a coding or non-coding sequence of the gene. The second RNA cancomprise for example an enhancer region, a tRNA, a RNA encoding anenhancer element, a RNA encoding a transcription factor, a micro RNA,stRNA, or other non-coding RNA that is involved in the expression of thetarget gene.

In one embodiment, the invention features a method for modulating theexpression of more than one gene within an organism comprising: (a)synthesizing a multifunctional siNA molecule of the invention, which canbe chemically-modified or unmodified, wherein the multifunctional siNAcomprises a first strand and a second strand that are complementary toeach other, and wherein the first strand comprises a region havingsequence complementarity to a RNA of a first gene or a portion thereofand the second strand comprises a region having sequence complementarityto a second gene or a portion thereof; and (b) introducing themultifunctional siNA molecule into the organism under conditionssuitable to modulate the expression of the genes in the organism. TheRNA of the first and second genes can independently comprise codingand/or non-coding sequences of the genes. In one embodiment, the firstgene encodes one or more cytokines and the second gene encodes one ormore receptors of the cytokine(s). In one embodiment, the first geneencodes one or more strains of a virus and the second gene encodes oneor strains of the same virus. In one embodiment, the first gene encodesone or more strains of a virus and the second gene encodes one orstrains of a different virus. In one embodiment, the first gene encodesone or more strains of a virus and the second gene encodes one or morecellular factors involved in infection or replication of the virus. Inone embodiment, the first gene encodes a first protein and the secondgene encodes a second protein that are involved in a common biologicpathway. In one embodiment, the first gene encodes a first protein andthe second gene encodes a second protein that are involved in divergentbiologic pathways.

In one embodiment, the invention features a method for modulating theexpression of one or more genes within a tissue or organ comprising: (a)synthesizing a multifunctional siNA molecule of the invention, which canbe chemically-modified or unmodified, wherein the multifunctional siNAcomprises sequences complementary to one or more RNAs of the gene(s) orportions thereof; and (b) introducing the multifunctional siNA moleculeinto the tissue or organ under conditions suitable to modulate theexpression of the gene(s) in the tissue or organ. In another embodiment,the tissue is ocular tissue and the organ is the eye. In anotherembodiment, the tissue comprises hepatocytes and/or hepatic tissue andthe organ is the liver.

In one embodiment, the invention features a method for modulating theexpression of a gene within a tissue or organ comprising: (a)synthesizing a multifunctional siNA molecule of the invention, which canbe chemically-modified or unmodified, wherein the multifunctional siNAcomprises a first strand and a second strand that are complementary toeach other, and wherein the first strand comprises a region havingsequence complementarity to a first portion of a RNA of the gene or aportion thereof and the second strand comprises a region having sequencecomplementarity to a second portion of the RNA of the gene or a portionthereof; and (b) introducing the multifunctional siNA molecule into thetissue or organ under conditions suitable to modulate the expression ofthe gene in the tissue or organ. The first and second portions of theRNA can comprise, for example, coding and/or non-coding sequences of thegene. In another embodiment, the tissue is ocular tissue and the organis the eye. In another embodiment, the tissue comprises hepatocytesand/or hepatic tissue and the organ is the liver.

In one embodiment, the invention features a method for modulating theexpression of more than one gene within a tissue or organ comprising:(a) synthesizing a multifunctional siNA molecule of the invention, whichcan be chemically-modified or unmodified, wherein the multifunctionalsiNA comprises a first strand and a second strand that are complementaryto each other, and wherein the first strand comprises a region havingsequence complementarity to a RNA of a first gene or a portion thereofand the second strand comprises a region having sequence complementarityto a RNA of a second gene or a portion thereof; and (b) introducing themultifunctional siNA molecule into the tissue or organ under conditionssuitable to modulate the expression of the genes in the tissue or organ.The RNA of the first and second genes can independently comprise codingand/or non-coding sequences of the genes. In one embodiment, the firstgene encodes one or more cytokines and the second gene encodes one ormore receptors of the cytokine(s). In one embodiment, the first geneencodes one or more strains of a virus and the second gene encodes oneor strains of the same virus. In one embodiment, the first gene encodesone or more strains of a virus and the second gene encodes one orstrains of a different virus. In one embodiment, the first gene encodesone or more strains of a virus and the second gene encodes one or morecellular factors involved in infection or replication of the virus. Inone embodiment, the first gene encodes a first protein and the secondgene encodes a second protein that are involved in a common biologicpathway. In one embodiment, the first gene encodes a first protein andthe second gene encodes a second protein that are involved in divergentbiologic pathways. In another embodiment, the tissue is ocular tissueand the organ is the eye. In another embodiment, the tissue compriseshepatocytes and/or hepatic tissue and the organ is the liver.

The multifunctional siNA molecules of the invention can be designed todown regulate or inhibit target gene expression in a biological systemby targeting of a variety of nucleic acid molecules (e.g., RNA). In oneembodiment, the multifunctional siNA molecules of the invention are usedto target various RNAs corresponding to a target gene. Non-limitingexamples of such RNAs include messenger RNA (mRNA), alternate RNA splicevariants of target gene(s), post-transcriptionally modified RNA oftarget gene(s), pre-mRNA of target gene(s), and/or RNA templates. Ifalternate splicing produces a family of transcripts that aredistinguished by usage of appropriate exons, the instant invention canbe used to inhibit gene expression through the appropriate exons tospecifically inhibit or to distinguish among the functions of genefamily members. For example, a protein that contains an alternativelyspliced transmembrane domain can be expressed in both membrane bound andsecreted forms. Use of the invention to target the exon containing thetransmembrane domain can be used to determine the functionalconsequences of pharmaceutical targeting of membrane bound as opposed tothe secreted form of the protein. Non-limiting examples of applicationsof the invention relating to targeting these RNA molecules includetherapeutic pharmaceutical applications, pharmaceutical discoveryapplications, molecular diagnostic and gene function applications, andgene mapping, for example using single nucleotide polymorphism mappingwith multifunctional siNA molecules of the invention. Such applicationscan be implemented using known gene sequences or from partial sequencesavailable from an expressed sequence tag (EST).

In another embodiment, the multifunctional siNA molecules of theinvention are used to target conserved sequences corresponding to a genefamily or gene families (e.g., different isoforms or different membersof a superfamily of genes, such as interleukin superfamily genes, tumornecrosis family superfamily genes, viral strains etc. (see for exampleMcSwiggen et al., WO 03/74654). As such, multifunctional siNA moleculestargeting multiple gene targets can provide increased therapeuticeffect. In addition, multifunctional siNA can be used to characterizepathways of gene function in a variety of applications. For example, thepresent invention can be used to inhibit the activity of target gene(s)in a pathway to determine the function of uncharacterized gene(s) ingene function analysis, mRNA function analysis, or translationalanalysis. The invention can be used to determine potential target genepathways involved in various diseases and conditions towardpharmaceutical development. The invention can be used to understandpathways of gene expression involved in, for example, in development,such as prenatal development and postnatal development, and/or theprogression and/or maintenance of cancer, infectious disease,autoimmunity, inflammation, endocrine disorders, renal disease, oculardisease, pulmonary disease, neurologic disease, cardiovascular disease,birth defects, aging, any other disease or condition related to geneexpression.

In one embodiment, multifunctional siNA molecule(s) and/or methods ofthe invention are used to down-regulate or inhibit the expression ofgene(s) that encode RNA referred to by Genbank Accession, for examplegenes encoding RNA sequence(s) referred to herein by Genbank Accessionnumber. See, for example, McSwiggen et al., WO 03/74654 incorporated byreference herein in its entirety for a list of mammalian and viraltargets.

In one embodiment, the invention features a method comprising: (a)generating a library of multifunctional siNA constructs having apredetermined complexity; and (b) assaying the multifunctional siNAconstructs of (a) above, under conditions suitable to determineaccessible target sites within the target RNA sequence. In oneembodiment, the multifunctional siNA molecules of (a) have strands of afixed length, for example, about 28 nucleotides in length. In anotherembodiment, the multifunctional siNA molecules of (a) are of differinglength, for example having strands of about 19 to about 34 (e.g., about19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34)nucleotides in length. The assay can comprise a cell culture system inwhich target RNA is expressed. In another embodiment, fragments oftarget RNA are analyzed for detectable levels of cleavage, for exampleby gel electrophoresis, northern blot analysis, or RNAse protectionassays, to determine the most suitable target site(s) within the targetRNA sequence. The target RNA sequence can be obtained as is known in theart, for example, by cloning and/or transcription for in vitro systems,and by cellular expression in in vivo systems.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising amultifunctional siNA molecule of the invention, which can bechemically-modified or unmodified, in a pharmaceutically acceptablecarrier or diluent. In another embodiment, the invention features apharmaceutical composition comprising multifunctional siNA molecules ofthe invention, which can be chemically-modified, targeting one or moregenes in a pharmaceutically acceptable carrier or diluent. In anotherembodiment, the invention features a method for diagnosing a disease orcondition in a subject comprising administering to the subject acomposition of the invention under conditions suitable for the diagnosisof the disease or condition in the subject. In another embodiment, theinvention features a method for treating or preventing a disease orcondition in a subject, comprising administering to the subject acomposition of the invention under conditions suitable for the treatmentor prevention of the disease or condition in the subject, alone or inconjunction with one or more other therapeutic compounds. In yet anotherembodiment, the invention features a method for reducing or preventingtissue rejection in a subject comprising administering to the subject acomposition of the invention under conditions suitable for the reductionor prevention of tissue rejection in the subject.

In another embodiment, the invention features a method for validating agene target in a biological system comprising: (a) synthesizing amultifunctional siNA molecule of the invention, which can bechemically-modified or unmodified, wherein the multifunctional siNAcomprises a first sequence and a second sequence that are complementaryto each other, and wherein the first sequence is complementary to afirst portion of a RNA of the gene or a portion thereof and the secondsequence is complementary a second portion of the RNA of the gene or aportion thereof; and (b) introducing the multifunctional siNA moleculeinto a cell, tissue, or organism under conditions suitable formodulating expression of the target gene in the cell, tissue, ororganism; and (c) determining the function of the gene by assaying forany phenotypic change in the cell, tissue, or organism.

In another embodiment, the invention features a method for validating abiologic pathway comprising two gene targets in a biological systemcomprising: (a) synthesizing a multifunctional siNA molecule of theinvention, which can be chemically-modified or unmodified, wherein themultifunctional siNA comprises a first sequence and a second sequencethat are complementary to each other, and wherein the first sequence iscomplementary to a RNA of a first gene or a portion thereof and thesecond sequence is complementary a RNA of a second gene or a portionthereof; and (b) introducing the multifunctional siNA molecule into acell, tissue, or organism under conditions suitable for modulatingexpression of the target genes in the cell, tissue, or organism; and (c)determining the function of the biologic pathway by assaying for anyphenotypic change in the cell, tissue, or organism.

In another embodiment, the invention features a method for validating abiologic pathway comprising two or more gene targets in a biologicalsystem comprising: (a) synthesizing a multifunctional siNA molecule ofthe invention, which can be chemically-modified or unmodified, whereinthe multifunctional siNA comprises a first sequence and a secondsequence that are complementary to each other, and wherein the firstsequence is complementary to a RNA of a one or more first gene targetsor a portion thereof and the second sequence is complementary a RNA ofone or more second gene targets or a portion thereof; and (b)introducing the multifunctional siNA molecule into a cell, tissue, ororganism under conditions suitable for modulating expression of thetarget genes in the cell, tissue, or organism; and (c) determining thefunction of the biologic pathway by assaying for any phenotypic changein the cell, tissue, or organism.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human,animal, plant, insect, bacterial, viral or other sources, wherein thesystem comprises the components required for biologic activity (e.g.,inhibition of gene expression). The term “biological system” includes,for example, a cell, tissue, or organism, or extract thereof.

By “phenotypic change” is meant any detectable change to a cell thatoccurs in response to contact or treatment with a nucleic acid moleculeof the invention (e.g., multifunctional siNA). Such detectable changesinclude, but are not limited to, changes in shape, size, proliferation,motility, protein expression or RNA expression or other physical orchemical changes as can be assayed by methods known in the art. Thedetectable change can also include expression of reportergenes/molecules such as Green Florescent Protein (GFP) or various tagsthat are used to identify an expressed protein or any other cellularcomponent that can be assayed.

In one embodiment, the invention features a kit containing amultifunctional siNA molecule of the invention, which can bechemically-modified or unmodified, that can be used to modulate theexpression of a target gene in biological system, including, forexample, in a cell, tissue, or organism. In one embodiment, theinvention features a kit containing a multifunctional siNA molecule ofthe invention, which can be chemically-modified or unmodified, that canbe used to modulate the expression of more than one target gene inbiological system, including, for example, in a cell, tissue, ororganism. In another embodiment, the invention features a kit containingmore than one multifunctional siNA molecule of the invention, which canbe chemically-modified, that can be used to modulate the expression ofmore than one target gene in a biological system, including, forexample, in a cell, tissue, or organism.

In one embodiment, the invention features a cell containing one or moremultifunctional siNA molecules of the invention, which can bechemically-modified or unmodified. In another embodiment, the cellcontaining a multifunctional siNA molecule of the invention is amammalian cell. In yet another embodiment, the cell containing amultifunctional siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of a multifunctional siNA duplexmolecule of the invention, which can be chemically-modified orunmodified, comprises: (a) synthesizing a self complementary nucleicacid sequence comprising nucleic acid molecule, defined herein asmultifunctional siNA molecule; (b) incubating the nucleic acid moleculeof (a) under conditions suitable for the multifunctional siNA moleculeto form a double-stranded multifunctional siNA molecule. In oneembodiment, synthesis of the self complementary nucleic acid sequencecontaining oligonucleotide or multifunctional siNA is by solid phaseoligonucleotide synthesis. In another embodiment the multifunctionalsiNA molecule is expressed from an expression vector or is enzymaticallysynthesized.

In another embodiment, the method of synthesis of multifunctional siNAmolecules of the invention comprises the teachings of Scaringe et al.,U.S. Pat. Nos. 5,889,136; 6,008,400; and 6,111,086, incorporated byreference herein in their entirety.

In one embodiment, the invention features a multifunctional siNAconstruct that mediates modulation or inhibition of gene expression in acell or reconstituted system, wherein the multifunctional siNA constructcomprises one or more chemical modifications, for example, one or morechemical modifications having any of Formulae III-IX or any combinationthereof that increases the nuclease resistance and/or overalleffectiveness or potency of the multifunctional siNA construct.

In another embodiment, the invention features a method for generatingmultifunctional siNA molecules with increased nuclease resistancecomprising (a) introducing nucleotides having any of Formula III-IX orany combination thereof into a multifunctional siNA molecule, and (b)assaying the multifunctional siNA molecule of step (a) under conditionssuitable for isolating multifunctional siNA molecules having increasednuclease resistance.

In another embodiment, the invention features a method for generatingmultifunctional siNA molecules with increased duration of effectcomprising (a) introducing nucleotides having any of Formula III-IX orany combination thereof into a multifunctional siNA molecule, and (b)assaying the multifunctional siNA molecule of step (a) under conditionssuitable for isolating multifunctional siNA molecules having increasedduration of effect.

In another embodiment, the invention features a method for generatingmultifunctional siNA molecules with increased delivery into a targetcell or tissue, such as hepatocytes, endothelial cells, T-cells, primarycells, and neuronal cells, comprising (a) introducing chemicalmodifications, conjugates, or nucleotides having any of Formula III-IXor any combination thereof into a multifunctional siNA molecule, and (b)assaying the multifunctional siNA molecule of step (a) under conditionssuitable for isolating multifunctional siNA molecules having increaseddelivery into a target cell or tissue. In one embodiment, the inventionfeatures multifunctional siNA duplex constructs that mediate modulationor inhibition of gene expression against a target gene, wherein themultifunctional siNA construct comprises one or more chemicalmodifications described herein that modulates the binding affinitybetween the two strands of the multifunctional siNA construct.

In one embodiment, the binding affinity between the strands of theduplex formed by the multifunctional siNA of the invention is modulatedto increase the activity of the multifunctional siNA molecule withregard to the ability of the multifunctional siNA to modulate geneexpression. In another embodiment the binding affinity between the twostrands of a multifunctional siNA duplex is decreased. The bindingaffinity between the strands of the multifunctional siNA construct canbe decreased by introducing one or more chemically modified nucleotidesin the multifunctional siNA sequence that disrupts the duplex stabilityof the multifunctional siNA (e.g., lowers the Tm of the duplex). Thebinding affinity between the strands of the multifunctional siNAconstruct can be decreased by introducing one or more nucleotides in themultifunctional siNA sequence that do not form Watson-Crick base pairs.The binding affinity between the strands of the multifunctional siNAconstruct can be decreased by introducing one or more wobble base pairsin the multifunctional siNA sequence. The binding affinity between thestrands of the multifunctional siNA construct can be decreased bymodifying the nucleobase composition of the multifunctional siNA, suchas by altering the G-C content of the multifunctional siNA sequence(e.g., decreasing the number of G-C base pairs in the multifunctionalsiNA sequence). These modifications and alterations in sequence can beintroduced selectively at pre-determined positions of themultifunctional siNA sequence to increase multifunctional siNA mediatedmodulation of gene expression. For example, such modifications andsequence alterations can be introduced to disrupt multifunctional siNAduplex stability between the 5′-end of one strand 3′-end of the otherstrand, the 3′-end of one strand and the 5′-end of the other strand, oralternately the middle of the multifunctional siNA duplex. In anotherembodiment, multifunctional siNA molecules are screened for optimizedactivity by introducing such modifications and sequence alterationseither by rational design based upon observed rules or trends inincreasing multifunctional siNA activity, or randomly via combinatorialselection processes that cover either partial or complete sequence spaceof the multifunctional siNA construct.

In another embodiment, the invention features a method for generating amultifunctional siNA duplex molecule with increased binding affinitybetween the strands of the multifunctional siNA molecule comprising (a)introducing nucleotides having any of Formula III-IX or any combinationthereof into a multifunctional siNA molecule, and (b) assaying themultifunctional siNA molecule of step (a) under conditions suitable forisolating a multifunctional siNA molecule having increased bindingaffinity between the strands of the multifunctional siNA molecule.

In one embodiment, the invention features a multifunctional siNAconstruct that modulates the expression of a target RNA, wherein themultifunctional siNA construct comprises one or more chemicalmodifications described herein that modulates the binding affinitybetween the multifunctional siNA construct and a complementary targetRNA sequence within a cell.

In one embodiment, the invention features a multifunctional siNAconstruct that modulates the expression of a target DNA, wherein themultifunctional siNA construct comprises one or more chemicalmodifications described herein that modulates the binding affinitybetween the multifunctional siNA construct and a complementary targetDNA sequence within a cell.

In another embodiment, the invention features a method for generating amultifunctional siNA molecule with increased binding affinity betweenthe multifunctional siNA molecule and a complementary target RNAsequence comprising (a) introducing nucleotides having any of FormulaIII-XI or any combination thereof into a multifunctional siNA molecule,and (b) assaying the multifunctional siNA molecule of step (a) underconditions suitable for isolating a multifunctional siNA molecule havingincreased binding affinity between the multifunctional siNA molecule anda complementary target RNA sequence.

In another embodiment, the invention features a method for generating amultifunctional siNA molecule with increased binding affinity betweenthe multifunctional siNA molecule and a complementary target DNAsequence comprising (a) introducing nucleotides having any of FormulaIII-IX or any combination thereof into a multifunctional siNA molecule,and (b) assaying the multifunctional siNA molecule of step (a) underconditions suitable for isolating a multifunctional siNA molecule havingincreased binding affinity between the multifunctional siNA molecule anda complementary target DNA sequence.

In one embodiment, the invention features a multifunctional siNAconstruct that modulates the expression of a target gene in a cell orreconstituted system, wherein the multifunctional siNA constructcomprises one or more chemical modifications described herein thatmodulates the cellular uptake of the multifunctional siNA construct.

In another embodiment, the invention features a method for generating amultifunctional siNA molecule against a target gene with improvedcellular uptake comprising (a) introducing nucleotides having any ofFormula III-IX or any combination thereof into a multifunctional siNAmolecule, and (b) assaying the multifunctional siNA molecule of step (a)under conditions suitable for isolating a multifunctional siNA moleculehaving improved cellular uptake.

In one embodiment, the invention features a multifunctional siNAconstruct that modulates the expression of a target gene, wherein themultifunctional siNA construct comprises one or more chemicalmodifications described herein that increases the bioavailability of themultifunctional siNA construct, for example, by attaching polymericconjugates such as polyethyleneglycol or equivalent conjugates thatimprove the pharmacokinetics of the multifunctional siNA construct, orby attaching conjugates that target specific tissue types or cell typesin vivo. Non-limiting examples of such conjugates are described inVargeese et al., U.S. Ser. No. 10/201,394 incorporated by referenceherein.

In one embodiment, the invention features a method for generating amultifunctional siNA molecule of the invention with improvedbioavailability comprising (a) introducing a conjugate into thestructure of a multifunctional siNA molecule, and (b) assaying themultifunctional siNA molecule of step (a) under conditions suitable forisolating multifunctional siNA molecules having improvedbioavailability. Such conjugates can include ligands for cellularreceptors, such as peptides derived from naturally occurring proteinligands; protein localization sequences, including cellular ZIP codesequences; antibodies; nucleic acid aptamers; vitamins and otherco-factors, such as folate and N-acetylgalactosamine; polymers, such aspolyethyleneglycol (PEG); phospholipids; cholesterol; polyamines, suchas spermine or spermidine; and others.

In one embodiment, the invention features a method for screeningmultifunctional siNA molecules against a target nucleic acid sequencecomprising, (a) generating a plurality of unmodified multifunctionalsiNA molecules, (b) assaying the multifunctional siNA molecules of step(a) under conditions suitable for isolating multifunctional siNAmolecules that are active in modulating expression of the target nucleicacid sequence, (c) optionally introducing chemical modifications (e.g.chemical modifications as described herein or as otherwise known in theart) into the active multifunctional siNA molecules of (b), and (d)optionally re-screening the chemically modified multifunctional siNAmolecules of (c) under conditions suitable for isolating chemicallymodified multifunctional siNA molecules that are active in modulatingexpression of the target nucleic acid sequence, for example in abiological system.

In one embodiment, the invention features a method for screeningmultifunctional siNA molecules against more than one target nucleic acidsequence comprising, (a) generating a plurality of unmodifiedmultifunctional siNA molecules, (b) assaying the multifunctional siNAmolecules of step (a) under conditions suitable for isolatingmultifunctional siNA molecules that are active in modulating expressionof the target nucleic acid sequences, (c) optionally introducingchemical modifications (e.g. chemical modifications as described hereinor as otherwise known in the art) into the active multifunctional siNAmolecules of (b), and (d) optionally re-screening the chemicallymodified multifunctional siNA molecules of (c) under conditions suitablefor isolating chemically modified multifunctional siNA molecules thatare active in modulating expression of the target nucleic acidsequences, for example in a biological system.

In one embodiment, the invention features a method for screeningmultifunctional siNA molecules against a target nucleic acid sequencecomprising (a) generating a plurality of chemically modifiedmultifunctional siNA molecules (e.g. multifunctional siNA molecules asdescribed herein or as otherwise known in the art), and (b) assaying themultifunctional siNA molecules of step (a) under conditions suitable forisolating chemically modified multifunctional siNA molecules that areactive in modulating expression of the target nucleic acid sequence.

In one embodiment, the invention features a method for screeningmultifunctional siNA molecules against more than one target nucleic acidsequence comprising (a) generating a plurality of chemically modifiedmultifunctional siNA molecules (e.g. multifunctional siNA molecules asdescribed herein or as otherwise known in the art), and (b) assaying themultifunctional siNA molecules of step (a) under conditions suitable forisolating chemically modified multifunctional siNA molecules that areactive in modulating expression of the target nucleic acid sequences.

In another embodiment, the invention features a method for generatingmultifunctional siNA molecules of the invention with improvedbioavailability comprising (a) introducing an excipient formulation to amultifunctional siNA molecule, and (b) assaying the multifunctional siNAmolecule of step (a) under conditions suitable for isolatingmultifunctional siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, nanoparticles, receptors, ligands,and others.

In another embodiment, the invention features a method for generating amultifunctional siNA molecule of the invention with improvedbioavailability comprising (a) introducing an excipient formulation to amultifunctional siNA molecule, and (b) assaying the multifunctional siNAmolecule of step (a) under conditions suitable for isolatingmultifunctional siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, and others.

In another embodiment, the invention features a method for generating amultifunctional siNA molecule of the invention with improvedbioavailability comprising (a) introducing nucleotides having any ofFormulae III-IX, a conjugate, or any combination thereof into amultifunctional siNA molecule, and (b) assaying the multifunctional siNAmolecule of step (a) under conditions suitable for isolatingmultifunctional siNA molecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to multifunctional siNA compounds of the present invention. Theattached PEG can be any molecular weight, preferably from about 2,000 toabout 50,000 daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include a multifunctional siNAmolecule of the invention and a vehicle that promotes introduction ofthe multifunctional siNA into cells of interest as described herein(e.g., using lipids and other methods of transfection known in the art,see for example Beigelman et al, U.S. Pat. No. 6,395,713). The kit canbe used, for example, for target validation, such as in determining genefunction and/or activity, in drug optimization, and in drug discovery(see for example Usman et al., U.S. Ser. No. 60/402,996). Such a kit canalso include instructions to allow a user of the kit to practice theinvention.

The term “multifunctional short interfering nucleic acid” or“multifunctional siNA” as used herein refers to any short interferingnucleic acid molecule comprising a first region of one strand havingnucleic acid sequence complementary to a first target nucleic acidsequence and a first region of the second strand having nucleic acidsequence complementary to a second target nucleic acid sequence, whereinthe first regions of each strand are not complementary to each other orif complementary then do not share more than 75% complementarity.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples ofsiNA molecules of the invention are shown in Beigelman et al. WO03/070918. For example the siNA can be a double-stranded polynucleotidemolecule comprising self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof and the sense region having nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof.The siNA can be assembled from two separate oligonucleotides, where onestrand is the sense strand and the other is the antisense strand,wherein the antisense and sense strands are self-complementary (i.e.each strand comprises nucleotide sequence that is complementary tonucleotide sequence in the other strand; such as where the antisensestrand and sense strand form a duplex or double stranded structure, forexample wherein the double stranded region is about 19 base pairs); theantisense strand comprises nucleotide sequence that is complementary tonucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense strand comprises nucleotide sequence correspondingto the target nucleic acid sequence or a portion thereof. Alternatively,the siNA is assembled from a single oligonucleotide, where theself-complementary sense and antisense regions of the siNA are linked bymeans of a nucleic acid based or non-nucleic acid-based linker(s). ThesiNA can be a polynucleotide with a duplex, asymmetric duplex, hairpinor asymmetric hairpin secondary structure, having self-complementarysense and antisense regions, wherein the antisense region comprisesnucleotide sequence that is complementary to nucleotide sequence in aseparate target nucleic acid molecule or a portion thereof and the senseregion having nucleotide sequence corresponding to the target nucleicacid sequence or a portion thereof. The siNA can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siNA molecule capable of mediating RNAi. The siNA canalso comprise a single stranded polynucleotide having nucleotidesequence complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof (for example, where such siNA moleculedoes not require the presence within the siNA molecule of nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof), wherein the single stranded polynucleotide can furthercomprise a terminal phosphate group, such as a 5′-phosphate (see forexample Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al.,2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic intercations, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing,translational inhibition, or epigenetics. For example, siNA molecules ofthe invention can be used to epigenetically silence genes at both thepost-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure to alter gene expression (see, for example,Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237).

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g.,multifunctional siNA) of the invention. In one embodiment, inhibition,down-regulation or reduction with an multifunctional siNA molecule isbelow that level observed in the presence of an inactive or attenuatedmolecule. In another embodiment, inhibition, down-regulation, orreduction with multifunctional siNA molecules is below that levelobserved in the presence of, for example, an multifunctional siNAmolecule with scrambled sequence or with mismatches. In anotherembodiment, inhibition, down-regulation, or reduction of gene expressionwith a nucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence.

By “palindrome” or “repeat” nucleic acid sequence is meant, a nucleicacid sequence whose 5′-to-3′ sequence is identical when present in aduplex. For example, a palindrome sequence of the invention in a duplexcan comprise sequence having the same sequence when one strand of theduplex is read in the 5′-to-3′ direction (left to right) and the otherstrand is read 3′-to-5′ direction (right to left). In another example, arepeat sequence of the invention can comprise a sequence having repeatednucleotides so arranged as to provide self complementarity (e.g. 5′-AUAU. . . -3′; 5′-AAUU . . . -3′; 5′-UAUA . . . -3′; 5′-UUAA . . . -3′;5′-CGCG . . . -3′; 5′-CCGG . . . -3′,5′-GGCC . . . -3′; 5′-CCGG . . .-3′; or any expanded repeat thereof etc.). The palindrome or repeatsequence can comprise about 2 to about 24 nucleotides in even numbers,(e.g., 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 nucleotides). Allthat is required of the palindrome or repeat sequence is that itcomprises nucleic acid sequence whose 5′-to-3′ sequence is identicalwhen present in a duplex, either alone or as part of a longer nucleicacid sequence. The palindrome or repeat sequence of the invention cancomprise chemical modifications as described herein that can form, forexample, Watson Crick or non-Watson Crick base pairs.

By “gene”, or “target gene”, is meant, a nucleic acid that encodes anRNA, for example, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide. A gene or target gene can alsoencode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as smalltemporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomalRNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Suchnon-coding RNAs can serve as target nucleic acid molecules formultifunctional siNA mediated RNA interference in modulating theactivity of fRNA or ncRNA involved in functional or regulatory cellularprocesses. Abberant fRNA or ncRNA activity leading to disease cantherefore be modulated by multifunctional siNA molecules of theinvention. multifunctional siNA molecules targeting fRNA and ncRNA canalso be used to manipulate or alter the genotype or phenotype of anorganism or cell, by intervening in cellular processes such as geneticimprinting, transcription, translation, or nucleic acid processing(e.g., transamination, methylation etc.). The target gene can be a genederived from a cell, an endogenous gene, a transgene, or exogenous genessuch as genes of a pathogen, for example a virus, which is present inthe cell after infection thereof. The cell containing the target genecan be derived from or contained in any organism, for example a plant,animal, protozoan, virus, bacterium, or fungus. Non-limiting examples ofplants include monocots, dicots, or gymnosperms. Non-limiting examplesof animals include vertebrates or invertebrates (see for example Zwicket al., U.S. Pat. No. 6,350,934, incorporated by reference herein).Non-limiting examples of fungi include molds or yeasts. Examples oftarget genes can be found generally in the art, see for exampleMcSwiggen et al., WO 03/74654 and Zwick et al., U.S. Pat. No. 6,350,934,incorporated by reference herein.

By “highly conserved sequence region” is meant, a nucleotide sequence ofone or more regions in a target gene does not vary significantly fromone generation to the other or from one biological system to the other.

By “cancer” is meant a group of diseases characterized by uncontrolledgrowth and/or spread of abnormal cells.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA, such as endogenous DNA or RNA, viral DNA or viral RNA, orother RNA encoded by a gene, virus, bacteria, fungus, mammal, or plant.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity or inhibition of gene expression or formation of doublestranded oligonucleotides by the multifunctional siNA molecules.Determination of binding free energies for nucleic acid molecules iswell known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant.Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). Apercent complementarity indicates the percentage of contiguous residuesin a nucleic acid molecule that can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in thefirst oligonuelcotide being based paired to a second nucleic acidsequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and100% complementary respectively). “Perfectly complementary” or “perfectcomplementarity” means that all the contiguous residues of a nucleicacid sequence will hydrogen bond with the same number of contiguousresidues in a second nucleic acid sequence.

The multifunctional siNA molecules of the invention represent a noveltherapeutic approach to a broad spectrum of diseases and conditions,including cancer or cancerous disease, infectious disease, oculardisease, cardiovascular disease, neurological disease, prion disease,inflammatory disease, autoimmune disease, pulmonary disease, renaldisease, liver disease, mitochondrial disease, endocrine disease,reproduction related diseases and conditions, and any other indicationsthat can respond to the level of an expressed gene product or a foreignnucleic acid, such as viral, fungal or bacterial genome, in a cell ororgansim.

In one embodiment of the present invention, each strand of amultifunctional siNA molecule of the invention is independently about 21to about 44 nucleotides in length, in specific embodiments about 21, 22,23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, or 44 nucleotides in length. In another embodiment, themultifunctional multifunctional siNA duplexes of the inventionindependently comprise about 17 to about 44 base pairs (e.g., about 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43 or 44 base pairs). Exemplarymultifunctional multifunctional siNA molecules of the invention areshown in FIGS. 1-4.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or hybrid, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

The multifunctional siNA molecules of the invention are added directly,or can be complexed with cationic lipids, packaged within liposomes, orotherwise delivered to target cells or tissues. The nucleic acid ornucleic acid complexes can be locally administered to relevant tissuesex vivo, or in vivo through injection, infusion pump or stent, with orwithout their incorporation in biopolymers.

In another aspect, the invention provides mammalian cells containing oneor more multifunctional siNA molecules of this invention. The one ormore multifunctional siNA molecules can independently be targeted to thesame or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribo-furanose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the multifunctionalsiNA or internally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter, that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intracellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise an acetyl orprotected acetyl group.

The term “thiophosphonoacetate” as used herein refers to aninternucleotide linkage having Formula I, wherein Z comprises an acetylor protected acetyl group and W comprises a sulfur atom or alternately Wcomprises an acetyl or protected acetyl group and Z comprises a sulfuratom.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar, for example where any of the ribosecarbons (C1, C2, C3, C4, or C5), are independently or in combinationabsent from the nucleotide.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to treatdiseases or conditions discussed herein (e.g., cancers and otherproliferative conditions, viral infection, inflammatory disease,autoimmunity, pulmonary disease, renal disease, ocular disease, etc.).For example, to treat a particular disease or condition, themultifunctional siNA molecules can be administered to a subject or canbe administered to other appropriate cells evident to those skilled inthe art, individually or in combination with one or more drugs underconditions suitable for the treatment.

In one embodiment, the invention features a method for treating orpreventing a disease or condition in a subject, wherein the disease orcondition is related to angiogenesis or neovascularization, comprisingadministering to the subject a multifunctional siNA molecule of theinvention under conditions suitable for the treatment or prevention ofthe disease or condition in the subject, alone or in conjunction withone or more other therapeutic compounds. In another embodiment, thedisease or condition resulting from angiogenesis, such as tumorangiogenesis leading to cancer, such as without limitation to breastcancer, lung cancer (including non-small cell lung carcinoma), prostatecancer, colorectal cancer, brain cancer, esophageal cancer, bladdercancer, pancreatic cancer, cervical cancer, head and neck cancer, skincancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma,renal cell carcinoma, gallbladder adeno carcinoma, parotidadenocarcinoma, ovarian cancer, melanoma, lymphoma, glioma, endometrialsarcoma, and multidrug resistant cancers, diabetic retinopathy, maculardegeneration, age related macular degeneration, macular adema,neovascular glaucoma, myopic degeneration, arthritis, psoriasis,endometriosis, female reproduction, verruca vulgaris, angiofibroma oftuberous sclerosis, pot-wine stains, Sturge Weber syndrome,Kippel-Trenaunay-Weber syndrome, Osler-Weber-Rendu syndrome, renaldisease such as Autosomal dominant polycystic kidney disease (ADPKD),restenosis, arteriosclerosis, and any other diseases or conditions thatare related to gene expression or will respond to RNA interference in acell or tissue, alone or in combination with other therapies.

In one embodiment, the invention features a method for treating orpreventing an ocular disease or condition in a subject, wherein theocular disease or condition is related to angiogenesis orneovascularization (such as those involving genes in the vascularendothelial growth factor, VEGF pathway or TGF-beta pathway), comprisingadministering to the subject a multifunctional siNA molecule of theinvention under conditions suitable for the treatment or prevention ofthe disease or condition in the subject, alone or in conjunction withone or more other therapeutic compounds. In another embodiment, theocular disease or condition comprises macular degeneration, age relatedmacular degeneration, diabetic retinopathy, macular adema, neovascularglaucoma, myopic degeneration, trachoma, scarring of the eye, cataract,ocular inflammation and/or ocular infections.

In one embodiment, the invention features a method of locallyadministering (e.g. by injection, such as intraocular, intratumoral,periocular, intracranial, etc., topical administration, catheter or thelike) to a tissue or cell (e.g., ocular or retinal, brain, CNS) a doublestranded RNA formed by a multifunctional siNA molecule or a vectorexpressing multifunctional siNA molecule, comprising nucleotide sequencethat is complementary to nucleotide sequence of target RNA, or a portionthereof, (e.g., target RNA encoding VEGF or a VEGF receptor) comprisingcontacting said tissue of cell with said double stranded RNA underconditions suitable for said local administration.

In one embodiment, the invention features a method of systemicallyadministering (e.g. by injection, such as subcutaneous, intravenous,topical administration, or the like) to a tissue or cell in a subject, adouble stranded RNA formed by a multifunctional siNA molecule or avector expressing multifunctional siNA molecule comprising nucleotidesequence that is complementary to nucleotide sequence of target RNA, ora portion thereof, (e.g., target RNA encoding VEGF or a VEGF receptor)comprising contacting said subject with said double stranded RNA underconditions suitable for said systemic administration.

In one embodiment, the invention features a method for treating orpreventing tumor angiogenesis in a subject comprising administering tothe subject a multifunctional siNA molecule of the invention underconditions suitable for the treatment or prevention of tumorangiogenesis in the subject, alone or in conjunction with one or moreother therapeutic compounds.

In one embodiment, the invention features a method for treating orpreventing viral infection or replication in a subject comprisingadministering to the subject a multifunctional siNA molecule of theinvention under conditions suitable for the treatment or prevention ofviral infection or replication in the subject, alone or in conjunctionwith one or more other therapeutic compounds.

In one embodiment, the invention features a method for treating orpreventing autoimmune disease in a subject comprising administering tothe subject a multifunctional siNA molecule of the invention underconditions suitable for the treatment or prevention of autoimmunedisease in the subject, alone or in conjunction with one or more othertherapeutic compounds.

In one embodiment, the invention features a method for treating orpreventing neurologic disease (e.g., Alzheimer's disease, Huntingtondisease, Parkinson disease, ALS, multiple sclerosis, epilepsy, etc.) ina subject comprising administering to the subject a multifunctional siNAmolecule of the invention under conditions suitable for the treatment orprevention of neurologic disease in the subject, alone or in conjunctionwith one or more other therapeutic compounds.

In one embodiment, the invention features a method for treating orpreventing inflammation in a subject comprising administering to thesubject a multifunctional siNA molecule of the invention underconditions suitable for the treatment or prevention of inflammation inthe subject, alone or in conjunction with one or more other therapeuticcompounds.

In a further embodiment, the multifunctional siNA molecules can be usedin combination with other known treatments to treat conditions ordiseases discussed above. For example, the described molecules could beused in combination with one or more known therapeutic agents to treat adisease or condition. Non-limiting examples of other therapeutic agentsthat can be readily combined with a multifunctional siNA molecule of theinvention are enzymatic nucleic acid molecules, allosteric nucleic acidmolecules, antisense, decoy, or aptamer nucleic acid molecules,antibodies such as monoclonal antibodies, small molecules, and otherorganic and/or inorganic compounds including metals, salts and ions.

In another aspect of the invention, multifunctional siNA molecules thatinteract with target RNA molecules and down-regulate gene encodingtarget RNA molecules (for example target RNA molecules referred to byGenbank Accession numbers herein) are expressed from transcription unitsinserted into DNA or RNA vectors. The recombinant vectors can be DNAplasmids or viral vectors. multifunctional siNA expressing viral vectorscan be constructed based on, but not limited to, adeno-associated virus,retrovirus, adenovirus, or alphavirus. The recombinant vectors capableof expressing the multifunctional siNA molecules can be delivered asdescribed herein, and persist in target cells. Alternatively, viralvectors can be used that provide for transient expression ofmultifunctional siNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the multifunctional siNAmolecules interact with target nucleic acids and down-regulate genefunction or expression. Delivery of multifunctional siNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from asubject followed by reintroduction into the subject, or by any othermeans that would allow for introduction into the desired target cell.

In one embodiment, the expression vector comprises a transcriptioninitiation region, a transcription termination region, and a geneencoding at least one multifunctional siNA. The gene can be operablylinked to the initiation region and the termination region, in a mannerwhich allows expression and/or delivery of the multifunctional siNA. Inanother embodiment, the expression vector can comprises a transcriptioninitiation region, a transcription termination region, an open readingframe and a gene encoding at least one multifunctional siNA, wherein thegene is operably linked to the 3′-end of the open reading frame. Thegene can be operably linked to the initiation region, the open readingframe and the termination region in a manner which allows expressionand/or delivery of the multifunctional siNA. In another embodiment, theexpression vector comprises a transcription initiation region, atranscription termination region, an intron, and a gene encoding atleast one multifunctional siNA. The gene can be operably linked to theinitiation region, the intron, and the termination region in a mannerwhich allows expression and/or delivery of the multifunctional siNA. Inyet another embodiment, the expression vector comprises a transcriptioninitiation region, a transcription termination region, an intron, anopen reading frame, and a gene encoding at least one multifunctionalsiNA, wherein the gene is operably linked to the 3′-end of the openreading frame. The gene can be operably linked to the initiation region,the intron, the open reading frame and the termination region in amanner which allows expression and/or delivery of the multifunctionalsiNA.

The expression vector can be derived from, for example, a retrovirus, anadenovirus, an adeno-associated virus, an alphavirus or a bacterialplasmid as well as other known vectors. The expression vector can beoperably linked to a RNA polymerase II promoter element or a RNApolymerase III promoter element. The RNA polymerase III promoter can bederived from, for example, a transfer RNA gene, a U6 small nuclear RNAgene, or a TRZ RNA gene. The multifunctional siNA transcript cancomprise a sequence at its 5′-end homologous to the terminal 27nucleotides encoded by the U6 small nuclear RNA gene. The library ofmultifunctional siNA constructs can be a multimer random library. Themultimer random library can comprise at least one multifunctional siNA.

The multifunctional siNA of the instant invention can be chemicallysynthesized, expressed from a vector, or enzymatically synthesized.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to produce, express and/or deliver a desired nucleic acid, such asthe multifunctional siNA molecule of the invention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences. FIG. 1A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the first andsecond complementary regions are situated at the 3′-ends of eachpolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 1B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first and second complementaryregions are situated at the 5′-ends of each polynucleotide sequence inthe multifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences.

FIG. 2 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences. FIG. 2A shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe second complementary region is situated at the 3′-end of thepolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 2B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first complementary region issituated at the 5′-end of the polynucleotide sequence in themultifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. In one embodiment,these multifunctional siNA constructs are processed in vivo or in vitroto generate multifunctional siNA constructs as shown in FIG. 1.

FIG. 3 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences and wherein the multifunctional siNA constructfurther comprises a self complementary, palindrome, or repeat region,thus enabling shorter bifuctional siNA constructs that can mediate RNAinterference against differing target nucleic acid sequences. FIG. 3Ashows a non-limiting example of a multifunctional siNA molecule having afirst region that is complementary to a first target nucleic acidsequence (complementary region 1) and a second region that iscomplementary to a second target nucleic acid sequence (complementaryregion 2), wherein the first and second complementary regions aresituated at the 3′-ends of each polynucleotide sequence in themultifunctional siNA, and wherein the first and second complementaryregions further comprise a self complementary, palindrome, or repeatregion. The dashed portions of each polynucleotide sequence of themultifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 3B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first and second complementary regions are situated at the 5′-endsof each polynucleotide sequence in the multifunctional siNA, and whereinthe first and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences.

FIG. 4 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences and wherein themultifunctional siNA construct further comprises a self complementary,palindrome, or repeat region, thus enabling shorter bifuctional siNAconstructs that can mediate RNA interference against differing targetnucleic acid sequences. FIG. 4A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the secondcomplementary region is situated at the 3′-end of the polynucleotidesequence in the multifunctional siNA, and wherein the first and secondcomplementary regions further comprise a self complementary, palindrome,or repeat region. The dashed portions of each polynucleotide sequence ofthe multifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 2B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first complementary region is situated at the 5′-end of thepolynucleotide sequence in the multifunctional siNA, and wherein thefirst and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. In one embodiment, these multifunctional siNA constructs areprocessed in vivo or in vitro to generate multifunctional siNAconstructs as shown in FIG. 3.

FIG. 5 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidmolecules, such as separate RNA molecules encoding differing proteins,for example a cytokine and its corresponding receptor, differing viralstrains, a virus and a cellular protein involved in viral infection orreplication, or differing proteins involved in a common or divergentbiologic pathway that is implicated in the maintenance of progression ofdisease. Each strand of the multifunctional siNA construct comprises aregion having complementarity to separate target nucleic acid molecules.The multifunctional siNA molecule is designed such that each strand ofthe siNA can be utilized by the RISC complex to initiate RNAinterference mediated cleavage of its corresponding target. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 6 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidsequences within the same target nucleic acid molecule, such asalternate coding regions of a RNA, coding and non-coding regions of aRNA, or alternate splice variant regions of a RNA. Each strand of themultifunctional siNA construct comprises a region having complementarityto the separate regions of the target nucleic acid molecule. Themultifunctional siNA molecule is designed such that each strand of thesiNA can be utilized by the RISC complex to initiate RNA interferencemediated cleavage of its corresponding target region. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 7 shows non-limiting examples of non-Watson Crick base pairs thatcan be utilized in generating artificial self complementary, palindrome,or repeat sequences for designing siNA molecules of the invention.

FIG. 8 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double-stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle-stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms whichrecognizes a target RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIG. 9 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention against degradation, including(1) [3-3′]-inverted deoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula III herein. In addition, the 2′-deoxy nucleotide shown 5′to the terminal modifications shown can be another modified orunmodified nucleotide or non-nucleotide described herein, for examplemodifications having any of Formulae III-IX herein or any combinationthereof.

FIG. 10 shows non-limiting examples of chemically modified terminalphosphate groups of the invention.

FIG. 11A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate siNA constructs. FIG. 11A:A DNA oligomer is synthesized with a 5′-restriction (R1) site sequencefollowed by a region having sequence identical to a predetermined targetsequence, wherein the sense region comprises, for example, about 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides (N) in length, andwhich is followed by a 3′-restriction site (R2) which is adjacent to aloop sequence of defined sequence (X). FIG. 11B: The synthetic constructis then extended by DNA polymerase to generate a hairpin structurehaving self-complementary sequence. FIG. 11C: The construct is processedby restriction enzymes specific to R1 and R2 to generate adouble-stranded DNA which is then inserted into an appropriate vectorfor expression in cells. The transcription cassette is designed suchthat a U6 promoter region flanks each side of the dsDNA which generatesthe strands of the siNA. Poly T termination sequences can be added tothe constructs to generate U overhangs in the resulting transcript.

DETAILED DESCRIPTION OF THE INVENTION Synthesis of Nucleic AcidMolecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences) are preferably used for exogenous delivery.The simple structure of these molecules increases the ability of thenucleic acid to invade targeted regions of protein and/or RNA structure.Exemplary molecules of the instant invention are chemically synthesized,and others can similarly be synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table VII outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick &Jackson Synthesis Grade acetonitrile is used directly from the reagentbottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made upfrom the solid obtained from American International Chemical, Inc.Alternately, for the introduction of phosphorothioate linkages, Beaucagereagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile)is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table VII outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mMI₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & JacksonSynthesis Grade acetonitrile is used directly from the reagent bottle.S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from thesolid obtained from American International Chemical, Inc. Alternately,for the introduction of phosphorothioate linkages, Beaucage reagent(3H-1,2-Benzodithiol-3-one 1,1-dioxide0.05 M in acetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10minutes. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO:1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperatureTEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and assembled together to form a duplex orjoined together post-synthetically, for example, by ligation (Moore etal., 1992, Science 256, 9923; Draper et al., International PCTpublication No. WO 93/23569; Shabarova et al., 1991, Nucleic AcidsResearch 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16,951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or byhybridization following synthesis and/or deprotection.

A siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein the two fragments comprise the same nucleicacid sequence and are self complementary.

siNA constructs can be purified by gel electrophoresis using generalmethods or can be purified by high pressure liquid chromatography (HPLC;see Wincott et al., supra, the totality of which is hereby incorporatedherein by reference) and re-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Alternatively, certain siNA molecules of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.Natl. Acad. Sci., USA 83, 399; Thompson et al., 1995, Nucleic AcidsRes., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45). Those skilledin the art realize that any nucleic acid can be expressed in eukaryoticcells from the appropriate DNA/RNA vector. The activity of such nucleicacids can be augmented by their release from the primary transcript by aenzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan etal., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27,15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura etal., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J.Biol. Chem., 269, 25856).

In another aspect of the invention, siNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Noonberg et al.,5,624,803; Thompson, U.S. Pat. Nos. 5,902,880 and 6,146,886). Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described above, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of nucleic acid molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the siNA molecule interactswith the target mRNA and generates an RNAi response. Delivery of siNAmolecule expressing vectors can be systemic, such as by intravenous orintra-muscular administration, by administration to target cellsex-planted from a subject followed by reintroduction into the subject,or by any other means that would allow for introduction into the desiredtarget cell (for a review see Couture et al., 1996, TIG., 12, 510).

In one aspect the invention features an expression vector comprising anucleic acid sequence encoding at least one siNA molecule of the instantinvention. The expression vector can encode the self complementary siNAsequence that can self assemble upon expression from the vector into aduplex oligonucleotide. The nucleic acid sequences encoding the siNAmolecules of the instant invention can be operably linked in a mannerthat allows expression of the siNA molecule (see for example Noonberg etal., 5,624,803; Thompson, U.S. Pat. Nos. 5,902,880 and 6,146,886; Paulet al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002,Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology,19, 500; and Novina et al., 2002, Nature Medicine, 8, 681-686).

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); and c) a nucleicacid sequence encoding at least one of the siNA molecules of the instantinvention, wherein said sequence is operably linked to said initiationregion and said termination region, in a manner that allows expressionand/or delivery of the siNA molecule. The vector can optionally includean open reading frame (ORF) for a protein operably linked on the 5′ sideor the 3′-side of the sequence encoding the siNA of the invention;and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol II promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gaoand Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993,Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10,4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al.,1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S. A, 90, 8000-4;Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,1993, Science, 262, 1566). More specifically, transcription units suchas the ones derived from genes encoding U6 small nuclear (snRNA),transfer RNA (tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al.,International PCT Publication No. WO 96/18736. The above siNAtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In another aspect, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one of the siNAmolecules of the invention, in a manner that allows expression of thatsiNA molecule. The expression vector comprises in one embodiment; a) atranscription initiation region; b) a transcription termination region;and c) a nucleic acid sequence encoding at least one strand of the siNAmolecule, wherein the sequence is operably linked to the initiationregion and the termination region in a manner that allows expressionand/or delivery of the siNA molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; and d) a nucleic acid sequence encoding atleast one strand of a siNA molecule, wherein the sequence is operablylinked to the 3′-end of the open reading frame and wherein the sequenceis operably linked to the initiation region, the open reading frame andthe termination region in a manner that allows expression and/ordelivery of the siNA molecule. In yet another embodiment, the expressionvector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; and d) a nucleic acidsequence encoding at least one siNA molecule, wherein the sequence isoperably linked to the initiation region, the intron and the terminationregion in a manner which allows expression and/or delivery of thenucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; and e) a nucleic acid sequenceencoding at least one strand of a siNA molecule, wherein the sequence isoperably linked to the 3′-end of the open reading frame and wherein thesequence is operably linked to the initiation region, the intron, theopen reading frame and the termination region in a manner which allowsexpression and/or delivery of the siNA molecule.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; Burgin et al., supra;and Beigelman et al., WO 03/70918, all of which are incorporated byreference herein). All of the above references describe various chemicalmodifications that can be made to the base, phosphate and/or sugarmoieties of the nucleic acid molecules described herein. Modificationsthat enhance their efficacy in cells, and removal of bases from nucleicacid molecules to shorten oligonucleotide synthesis times and reducechemical requirements are desired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-β-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Usman and Cedergren, Trends in Biochem.Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No.WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995,J. Biol. Chem., 270, 25702; Beigelman et al., International PCTpublication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824;Beigelman et al., WO 03/70918; Usman et al., U.S. Pat. No. 5,627,053;;Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20,1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw andGait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma andEckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al.,1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are herebyincorporated in their totality by reference herein). Such publicationsdescribe general methods and strategies to determine the location ofincorporation of sugar, base and/or phosphate modifications and the likeinto nucleic acid molecules without modulating catalysis, and areincorporated by reference herein. In view of such teachings, similarmodifications can be used as described herein to modify the siNA nucleicacid molecules of the instant invention so long as the ability of siNAto promote RNAi is cells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

siNA molecules having chemical modifications that maintain or enhanceactivity are provided. Such a nucleic acid is also generally moreresistant to nucleases than an unmodified nucleic acid. Accordingly, thein vitro and/or in vivo activity should not be significantly lowered. Incases in which modulation is the goal, therapeutic nucleic acidmolecules delivered exogenously should optimally be stable within cellsuntil translation of the target RNA has been modulated long enough toreduce the levels of the undesirable protein. This period of time variesbetween hours to days depending upon the disease state. Improvements inthe chemical synthesis of RNA and DNA (Wincott et al., 1995, NucleicAcids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211,3-19 (incorporated by reference herein)) have expanded the ability tomodify nucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226, and McSwiggen et al., WO 03/70918).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention (see for example WO WO 02/094185 and U.S.Ser. No. 10/427,160 both incorporated by reference herein in theirentirety including the drawings). The present invention encompasses thedesign and synthesis of novel conjugates and complexes for the deliveryof molecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample, proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The present invention features compositions and conjugates to facilitatedelivery of molecules into a biological system such as cells. Theconjugates provided by the instant invention can impart therapeuticactivity by transferring therapeutic compounds across cellularmembranes. The present invention encompasses the design and synthesis ofnovel agents for the delivery of molecules, including but not limited tosiNA molecules. In general, the transporters described are designed tobe used either individually or as part of a multi-component system. Thecompounds of the invention generally shown in Formulae herein areexpected to improve delivery of molecules into a number of cell typesoriginating from different tissues, in the presence or absence of serum.

In another embodiment, the compounds of the invention are provided as asurface component of a lipid aggregate, such as a liposome encapsulatedwith the predetermined molecule to be delivered. Liposomes, which can beunilamellar or multilamellar, can introduce encapsulated material into acell by different mechanisms. For example, the liposome can directlyintroduce its encapsulated material into the cell cytoplasm by fusingwith the cell membrane. Alternatively, the liposome can becompartmentalized into an acidic vacuole (i.e., an endosome) and itscontents released from the liposome and out of the acidic vacuole intothe cellular cytoplasm.

In one embodiment the invention features a lipid aggregate formulationof the compounds described herein, including phosphatidylcholine (ofvarying chain length; e.g., egg yolk phosphatidylcholine), cholesterol,a cationic lipid, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polythyleneglycol-2000(DSPE-PEG2000). The cationic lipid component of this lipid aggregate canbe any cationic lipid known in the art such as dioleoyl1,2,-diacyl-3-trimethylammonium-propane (DOTAP). In another embodimentthis cationic lipid aggregate comprises a covalently bound compounddescribed in any of the Formulae herein.

In another embodiment, polyethylene glycol (PEG) is covalently attachedto the compounds of the present invention. The attached PEG can be anymolecular weight but is preferably between 2000-50,000 daltons.

The compounds and methods of the present invention are useful forintroducing nucleotides, nucleosides, nucleic acid molecules, lipids,peptides, proteins, and/or non-nucleosidic small molecules into a cell.For example, the invention can be used for nucleotide, nucleoside,nucleic acid, lipids, peptides, proteins, and/or non-nucleosidic smallmolecule delivery where the corresponding target site of action existsintracellularly.

In one embodiment, the compounds of the instant invention provideconjugates of molecules that can interact with cellular receptors, suchas high affinity folate receptors and ASGPr receptors, and provide anumber of features that allow the efficient delivery and subsequentrelease of conjugated compounds across biological membranes. Thecompounds utilize chemical linkages between the receptor ligand and thecompound to be delivered of length that can interact preferentially withcellular receptors. Furthermore, the chemical linkages between theligand and the compound to be delivered can be designed as degradablelinkages, for example by utilizing a phosphate linkage that is proximalto a nucleophile, such as a hydroxyl group. Deprotonation of thehydroxyl group or an equivalent group, as a result of pH or interactionwith a nuclease, can result in nucleophilic attack of the phosphateresulting in a cyclic phosphate intermediate that can be hydrolyzed.This cleavage mechanism is analogous RNA cleavage in the presence of abase or RNA nuclease. Alternately, other degradable linkages can beselected that respond to various factors such as UV irradiation,cellular nucleases, pH, temperature etc. The use of degradable linkagesallows the delivered compound to be released in a predetermined system,for example in the cytoplasm of a cell, or in a particular cellularorganelle.

The present invention also provides ligand derived phosphoramidites thatare readily conjugated to compounds and molecules of interest.Phosphoramidite compounds of the invention permit the direct attachmentof conjugates to molecules of interest without the need for usingnucleic acid phosphoramidite species as scaffolds. As such, the used ofphosphoramidite chemistry can be used directly in coupling the compoundsof the invention to a compound of interest, without the need for othercondensation reactions, such as condensation of the ligand to an aminogroup on the nucleic acid, for example at the N6 position of adenosineor a 2′-deoxy-2′-amino function. Additionally, compounds of theinvention can be used to introduce non-nucleic acid based conjugatedlinkages into oligonucleotides that can provide more efficient couplingduring oligonucleotide synthesis than the use of nucleic acid-basedphosphoramidites. This improved coupling can take into account improvedsteric considerations of abasic or non-nucleosidic scaffolds bearingpendant alkyl linkages.

Compounds of the invention utilizing triphosphate groups can be utilizedin the enzymatic incorporation of conjugate molecules intooligonucleotides. Such enzymatic incorporation is useful when conjugatesare used in post-synthetic enzymatic conjugation or selection reactions,(see for example Matulic-Adamic et al., 2000, Bioorg. Med. Chem. Lett.,10, 1299-1302; Lee et al., 2001, NAR., 29, 1565-1573; Joyce, 1989, Gene,82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992,Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268;Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS17,89-93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op.Biotech., 7, 442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94,4262; Tang et al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra;Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish etal., 1997, Biochemistry 36, 6495; Kuwabara et al., 2000, Curr. Opin.Chem. Biol., 4, 669).

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to a siNA molecule of the invention or thestrands of a siNA molecule of the invention. The biodegradable linker isdesigned such that its stability can be modulated for a particularpurpose, such as delivery to a particular tissue or cell type. Thestability of a nucleic acid-based biodegradable linker molecule can bemodulated by using various chemistries, for example combinations ofribonucleotides, deoxyribonucleotides, and chemically-modifiednucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino,2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modifiednucleotides. The biodegradable nucleic acid linker molecule can be adimer, trimer, tetramer or longer nucleic acid molecule, for example, anoligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 nucleotides in length, or can comprise a singlenucleotide with a phosphorus-based linkage, for example, aphosphoramidate or phosphodiester linkage. The biodegradable nucleicacid linker molecule can also comprise nucleic acid backbone, nucleicacid sugar, or nucleic acid base modifications (see for exampleMcSwiggen et al., WO 03/70918 and Vargeese et al., U.S. Ser. No.10/201,394 and 10/427,160).

The term “biodegradable” as used herein, refers to degradation in abiological system, for example enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein, refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

The term “alkyl” as used herein refers to a saturated aliphatichydrocarbon, including straight-chain, branched-chain “isoalkyl”, andcyclic alkyl groups. The term “alkyl” also comprises alkoxy, alkyl-thio,alkyl-thio-alkyl, alkoxyalkyl, alkylamino, alkenyl, alkynyl, alkoxy,cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl,C1-C6 hydrocarbyl, aryl or substituted aryl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably it is a lower alkyl offrom about 1 to about 7 carbons, more preferably about 1 to about 4carbons. The alkyl group can be substituted or unsubstituted. Whensubstituted the substituted group(s) preferably comprise hydroxy, oxy,thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl,alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl,cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6hydrocarbyl, aryl or substituted aryl groups. The term “alkyl” alsoincludes alkenyl groups containing at least one carbon-carbon doublebond, including straight-chain, branched-chain, and cyclic groups.Preferably, the alkenyl group has about 2 to about 12 carbons. Morepreferably it is a lower alkenyl of from about 2 to about 7 carbons,more preferably about 2 to about 4 carbons. The alkenyl group can besubstituted or unsubstituted. When substituted the substituted group(s)preferably comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy,alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl,alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substitutedaryl groups. The term “alkyl” also includes alkynyl groups containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group hasabout 2 to about 12 carbons. More preferably it is a lower alkynyl offrom about 2 to about 7 carbons, more preferably about 2 to about 4carbons. The alkynyl group can be substituted or unsubstituted. Whensubstituted the substituted group(s) preferably comprise hydroxy, oxy,thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl,alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl,cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6hydrocarbyl, aryl or substituted aryl groups. Alkyl groups or moietiesof the invention can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. The preferred substituent(s)of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano,alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” grouprefers to an alkyl group (as described above) covalently joined to anaryl group (as described above). Carbocyclic aryl groups are groupswherein the ring atoms on the aromatic ring are all carbon atoms. Thecarbon atoms are optionally substituted. Heterocyclic aryl groups aregroups having from about 1 to about 3 heteroatoms as ring atoms in thearomatic ring and the remainder of the ring atoms are carbon atoms.Suitable heteroatoms include oxygen, sulfur, and nitrogen, and includefuranyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl,pyrazinyl, imidazolyl and the like, all optionally substituted. An“amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl,alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R iseither alkyl, aryl, alkylaryl or hydrogen.

The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether,for example, methoxyethyl or ethoxymethyl.

The term “alkyl-thio-alkyl” as used herein refers to an alkyl-S-alkylthioether, for example, methylthiomethyl or methylthioethyl.

The term “amino” as used herein refers to a nitrogen containing group asis known in the art derived from ammonia by the replacement of one ormore hydrogen radicals by organic radicals. For example, the terms“aminoacyl” and “aminoalkyl” refer to specific N-substituted organicradicals with acyl and alkyl substituent groups respectively.

The term “alkenyl” as used herein refers to a straight or branchedhydrocarbon of a designed number of carbon atoms containing at least onecarbon-carbon double bond. Examples of “alkenyl” include vinyl, allyl,and 2-methyl-3-heptene.

The term “alkoxy” as used herein refers to an alkyl group of indicatednumber of carbon atoms attached to the parent molecular moiety throughan oxygen bridge. Examples of alkoxy groups include, for example,methoxy, ethoxy, propoxy and isopropoxy.

The term “alkynyl” as used herein refers to a straight or branchedhydrocarbon of a designed number of carbon atoms containing at least onecarbon-carbon triple bond. Examples of “alkynyl” include propargyl,propyne, and 3-hexyne.

The term “aryl” as used herein refers to an aromatic hydrocarbon ringsystem containing at least one aromatic ring. The aromatic ring canoptionally be fused or otherwise attached to other aromatic hydrocarbonrings or non-aromatic hydrocarbon rings. Examples of aryl groupsinclude, for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthaleneand biphenyl. Preferred examples of aryl groups include phenyl andnaphthyl.

The term “cycloalkenyl” as used herein refers to a C3-C8 cyclichydrocarbon containing at least one carbon-carbon double bond. Examplesof cycloalkenyl include cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl,cycloheptatrienyl, and cyclooctenyl.

The term “cycloalkyl” as used herein refers to a C3-C8 cyclichydrocarbon. Examples of cycloalkyl include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The term “cycloalkylalkyl,” as used herein, refers to a C3-C7 cycloalkylgroup attached to the parent molecular moiety through an alkyl group, asdefined above. Examples of cycloalkylalkyl groups includecyclopropylmethyl and cyclopentylethyl.

The terms “halogen” or “halo” as used herein refers to indicatefluorine, chlorine, bromine, and iodine.

The term “heterocycloalkyl,” as used herein refers to a non-aromaticring system containing at least one heteroatom selected from nitrogen,oxygen, and sulfur. The heterocycloalkyl ring can be optionally fused toor otherwise attached to other heterocycloalkyl rings and/ornon-aromatic hydrocarbon rings. Preferred heterocycloalkyl groups havefrom 3 to 7 members. Examples of heterocycloalkyl groups include, forexample, piperazine, morpholine, piperidine, tetrahydrofuran,pyrrolidine, and pyrazole. Preferred heterocycloalkyl groups includepiperidinyl, piperazinyl, morpholinyl, and pyrrolidinyl.

The term “heteroaryl” as used herein refers to an aromatic ring systemcontaining at least one heteroatom selected from nitrogen, oxygen, andsulfur. The heteroaryl ring can be fused or otherwise attached to one ormore heteroaryl rings, aromatic or non-aromatic hydrocarbon rings orheterocycloalkyl rings. Examples of heteroaryl groups include, forexample, pyridine, furan, thiophene, 5,6,7,8-tetrahydroisoquinoline andpyrimidine. Preferred examples of heteroaryl groups include thienyl,benzothienyl, pyridyl, quinolyl, pyrazinyl, pyrimidyl, imidazolyl,benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl,isoxazolyl, oxadiazolyl, isothiazolyl, benzisothiazolyl, triazolyl,tetrazolyl, pyrrolyl, indolyl, pyrazolyl, and benzopyrazolyl.

The term “C1-C6 hydrocarbyl” as used herein refers to straight,branched, or cyclic alkyl groups having 1-6 carbon atoms, optionallycontaining one or more carbon-carbon double or triple bonds. Examples ofhydrocarbyl groups include, for example, methyl, ethyl, propyl,isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl,neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, vinyl, 2-pentene,cyclopropylmethyl, cyclopropyl, cyclohexylmethyl, cyclohexyl andpropargyl. When reference is made herein to C1-C6 hydrocarbyl containingone or two double or triple bonds it is understood that at least twocarbons are present in the alkyl for one double or triple bond, and atleast four carbons for two double or triple bonds.

The term “phosphorus containing group” as used herein, refers to achemical group containing a phosphorus atom. The phosphorus atom can betrivalent or pentavalent, and can be substituted with O, H, N, S, C orhalogen atoms. Examples of phosphorus containing groups of the instantinvention include but are not limited to phosphorus atoms substitutedwith O, H, N, S, C or halogen atoms, comprising phosphonate,alkylphosphonate, phosphate, diphosphate, triphosphate, pyrophosphate,phosphorothioate, phosphorodithioate, phosphoramidate, phosphoramiditegroups, nucleotides and nucleic acid molecules.

The term “degradable linker” as used herein, refers to linker moietiesthat are capable of cleavage under various conditions. Conditionssuitable for cleavage can include but are not limited to pH, UVirradiation, enzymatic activity, temperature, hydrolysis, elimination,and substitution reactions, and thermodynamic properties of the linkage.

The term “photolabile linker” as used herein, refers to linker moietiesas are known in the art, that are selectively cleaved under particularUV wavelengths. Compounds of the invention containing photolabilelinkers can be used to deliver compounds to a target cell or tissue ofinterest, and can be subsequently released in the presence of a UVsource.

The term “nucleic acid conjugates” as used herein, refers to nucleoside,nucleotide and oligonucleotide conjugates.

The term “lipid” as used herein, refers to any lipophilic compound.Non-limiting examples of lipid compounds include fatty acids and theirderivatives, including straight chain, branched chain, saturated andunsaturated fatty acids, carotenoids, terpenes, bile acids, andsteroids, including cholesterol and derivatives or analogs thereof.

The term “folate” as used herein, refers to analogs and derivatives offolic acid, for example antifolates, dihydrofloates, tetrahydrofolates,tetrahydrorpterins, folinic acid, pteropolyglutamic acid, 1-deza,3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10 dideaza,8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroic acidderivatives.

The term “compounds with neutral charge” as used herein, refers tocompositions which are neutral or uncharged at neutral or physiologicalpH. Examples of such compounds are cholesterol and other steroids,cholesteryl hemisuccinate (CHEMS), dioleoyl phosphatidyl choline,distearoylphosphotidyl choline (DSPC), fatty acids such as oleic acid,phosphatidic acid and its derivatives, phosphatidyl serine, polyethyleneglycol-conjugated phosphatidylamine, phosphatidylcholine,phosphatidylethanolamine and related variants, prenylated compoundsincluding farnesol, polyprenols, tocopherol, and their modified forms,diacylsuccinyl glycerols, fusogenic or pore forming peptides,dioleoylphosphotidylethanolamine (DOPE), ceramide and the like.

The term “lipid aggregate” as used herein refers to a lipid-containingcomposition wherein the lipid is in the form of a liposome, micelle(non-lamellar phase) or other aggregates with one or more lipids.

The term “nitrogen containing group” as used herein refers to anychemical group or moiety comprising a nitrogen or substituted nitrogen.Non-limiting examples of nitrogen containing groups include amines,substituted amines, amides, alkylamines, amino acids such as arginine orlysine, polyamines such as spermine or spermidine, cyclic amines such aspyridines, pyrimidines including uracil, thymine, and cytosine,morpholines, phthalimides, and heterocyclic amines such as purines,including guanine and adenine.

Therapeutic nucleic acid molecules (e.g., siNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes; nucleic acid molecules coupled with known smallmolecule modulators; or intermittent treatment with combinations ofmolecules, including different motifs and/or other chemical orbiological molecules). The treatment of subjects with siNA molecules canalso include combinations of different types of nucleic acid molecules,such as enzymatic nucleic acid molecules (ribozymes), allozymes,antisense, 2,5-A oligoadenylate, decoys, and aptamers.

In another aspect a siNA molecule of the invention comprises one or more3′-cap structures.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, and Beigelman et al.,WO 03/70918 incorporated by reference herein). These terminalmodifications protect the nucleic acid molecule from exonucleasedegradation, and can help in delivery and/or localization within a cell.The cap can be present at the 3′-terminus of one or both strands of themultifunctional siNA (3′-cap). Non-limiting examples of the 3′-capinclude, but are not limited to, glyceryl, inverted deoxy a basicresidue (moiety), 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate;3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

By “nucleotide” as used herein is as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,S-alkylcytidines (e.g., 5-methylcytidine), S-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphonoacetate, and/or thiophosphonoacetate,phosphorodithioate, methylphosphonate, phosphotriester, morpholino,amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate,sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl,substitutions. For a review of oligonucleotide backbone modifications,see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis andProperties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker etal., 1994, Novel Backbone Replacements for Oligonucleotides, inCarbohydrate Modifications in Antisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1′ carbon ofβ-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-V11 and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′—NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878, which are both incorporated by reference in theirentireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

A siNA molecule of the invention can be adapted for use to treat anydisease, infection or condition associated with gene expression, andother indications that can respond to the level of gene product in acell or tissue, alone or in combination with other therapies. Forexample, a siNA molecule can comprise a delivery vehicle, includingliposomes, for administration to a subject, carriers and diluents andtheir salts, and/or can be present in pharmaceutically acceptableformulations. Methods for the delivery of nucleic acid molecules aredescribed in Akhtar et al., 1992, Trends Cell Bio., 2, 139; DeliveryStrategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995,Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang,1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACSSymp. Ser., 752, 184-192, all of which are incorporated herein byreference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan etal., PCT WO 94/02595 further describe the general methods for deliveryof nucleic acid molecules. These protocols can be utilized for thedelivery of virtually any nucleic acid molecule. Nucleic acid moleculescan be administered to cells by a variety of methods known to those ofskill in the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as biodegradable polymers, hydrogels, cyclodextrins (see forexample Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wanget al., International PCT publication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. US 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). In one embodiment,nucleic acid molecules or the invention are administered viabiodegradable implant materials, such as elastic shape memory polymers(see for example Lendelein and Langer, 2002, Science, 296, 1673).Alternatively, the nucleic acid/vehicle combination is locally deliveredby direct injection or by use of an infusion pump. Direct injection ofthe nucleic acid molecules of the invention, whether subcutaneous,intramuscular, or intradermal, can take place using standard needle andsyringe methodologies, or by needle-free technologies such as thosedescribed in Conry et al., 1999, Clin. Cancer Res., 5, 2330-2337 andBarry et al., International PCT Publication No. WO 99/31262. Manyexamples in the art describe CNS delivery methods of oligonucleotides byosmotic pump, (see Chun et al., 1998, Neuroscience Letters, 257,135-138, D'Aldin et al., 1998, Mol. Brain. Research, 55, 151-164, Drydenet al., 1998, J. Endocrinol., 157, 169-175, Ghirnikar et al., 1998,Neuroscience Letters, 247, 21-24) or direct infusion (Broaddus et al.,1997, Neurosurg. Focus, 3, article 4). Other routes of delivery include,but are not limited to oral (tablet or pill form) and/or intrathecaldelivery (Gold, 1997, Neuroscience, 76, 1153-1158). More detaileddescriptions of nucleic acid delivery and administration are provided inSullivan et al., supra, Draper et al., PCT WO93/23569, Beigelman et al.,PCT WO99/05094, and Klimuk et al., PCT WO99/04819 all of which have beenincorporated by reference herein. The molecules of the instant inventioncan be used as pharmaceutical agents. Pharmaceutical agents prevent,modulate the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state in a subject.

In addition, the invention features the use of methods to deliver thenucleic acid molecules of the instant invention to hematopoietic cells,including monocytes and lymphocytes. These methods are described indetail by Hartmann et al., 1998, J. Phamacol. Exp. Ther., 285(2),920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862; Filion andPhillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei,1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al., 1994, NucleicAcids Research, 22(22), 4681-8. Such methods, as described above,include the use of free oligonucleotide, cationic lipid formulations,liposome formulations including pH sensitive liposomes andimmunoliposomes, and bioconjugates including oligonucleotides conjugatedto fusogenic peptides, for the transfection of hematopoietic cells witholigonucleotides.

In one embodiment, a compound, molecule, or composition for thetreatment of ocular conditions (e.g., macular degeneration, diabeticretinopathy etc.) is administered to a subject intraocularly or byintraocular means. In another embodiment, a compound, molecule, orcomposition for the treatment of ocular conditions (e.g., maculardegeneration, diabetic retinopathy etc.) is administered to a subjectperiocularly or by periocular means (see for example Ahlheim et al.,International PCT publication No. WO 03/24420). In one embodiment, asiNA molecule and/or formulation or composition thereof is administeredto a subject intraocularly or by intraocular means. In anotherembodiment, a siNA molecule and/or formulation or composition thereof isadministered to a subject periocularly or by periocular means.Periocular administration generally provides a less invasive approach toadministering siNA molecules and formulation or composition thereof to asubject (see for example Ahlheim et al., International PCT publicationNo. WO 03/24420). The use of periocular administration also minimizesthe risk of retinal detachment, allows for more frequent dosing oradministration, provides a clinically relevant route of administrationfor macular degeneration and other optic conditions, and also providesthe possibility of using reservoirs (e.g., implants, pumps or otherdevices) for drug delivery.

In one embodiment, a siNA molecule of the invention is complexed withmembrane disruptive agents such as those described in U.S. PatentApplication Publication No. 20010007666, incorporated by referenceherein in its entirety including the drawings. In another embodiment,the membrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310, incorporated by referenceherein in its entirety including the drawings.

In one embodiment, siNA molecules of the invention are formulated orcomplexed with polyethylenimine (e.g., linear or branched PEI) and/orpolyethylenimine derivatives, including for example grafted PEIs such asgalactose PEI, cholesterol PEI, antibody derivatized PEI, andpolyethylene glycol PEI (PEG-PEI) derivatives thereof (see for exampleOgris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003,Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, PharmaceuticalResearch, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22,46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Petersonet al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999,Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNASUSA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release,60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; andSagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, a siNA molecule of the invention comprises abioconjugate, for example a nucleic acid conjugate as described inVargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S.Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat. No. 6,235,886;U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S. Pat. No.5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedinto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as tablets, capsules orelixirs for oral administration, suppositories for rectaladministration, sterile solutions, suspensions for injectableadministration, and the other compositions known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or subject, including for example a human.Suitable forms, in part, depend upon the use or the route of entry, forexample oral, transdermal, or by injection. Such forms should notprevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption oraccumulation of drugs in the blood stream followed by distributionthroughout the entire body. Administration routes that lead to systemicabsorption include, without limitation: intravenous, subcutaneous,intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.Each of these administration routes exposes the siNA molecules of theinvention to an accessible diseased tissue. The rate of entry of a druginto the circulation has been shown to be a function of molecular weightor size. The use of a liposome or other drug carrier comprising thecompounds of the instant invention can potentially localize the drug,for example, in certain tissue types, such as the tissues of thereticular endothelial system (RES). A liposome formulation that canfacilitate the association of drug with the surface of cells, such as,lymphocytes and macrophages is also useful. This approach can provideenhanced delivery of the drug to target cells by taking advantage of thespecificity of macrophage and lymphocyte immune recognition of abnormalcells, such as cancer cells.

By “pharmaceutically acceptable formulation” is meant a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity. Non-limiting examples of agentssuitable for formulation with the nucleic acid molecules of the instantinvention include: P-glycoprotein inhibitors (such as Pluronic P85),which can enhance entry of drugs into the CNS (Jolliet-Riant andTillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery after intracerebral implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge,Mass.); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate, which can deliver drugs across the blood brainbarrier and can alter neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Othernon-limiting examples of delivery strategies for the nucleic acidmolecules of the instant invention include material described in Boadoet al., 1998J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBSLett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596;Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada etal., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999,PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995,95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995,Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable foradministering nucleic acid molecules of the invention to specific celltypes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu,1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and bindsbranched galactose-terminal glycoproteins, such as asialoorosomucoid(ASOR). In another example, the folate receptor is overexpressed in manycancer cells. Binding of such glycoproteins, synthetic glycoconjugates,or folates to the receptor takes place with an affinity that stronglydepends on the degree of branching of the oligosaccharide chain, forexample, triatennary structures are bound with greater affinity thanbiatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22,611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee andLee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose,galactosamine, or folate based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto, for example, the treatment of liver disease, cancers of the liver,or other cancers. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of nucleicacid bioconjugates of the invention. Non-limiting examples of suchbioconjugates are described in Vargeese et al., U.S. Ser. No.10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser.No. 10/151,116, filed May 17, 2002. In one embodiment, nucleic acidmolecules of the invention are complexed with or covalently attached tonanoparticles, such as Hepatitis B virus S, M, or L evelope proteins(see for example Yamado et al., 2003, Nature Biotechnology, 21, 885). Inone embodiment, nucleic acid molecules of the invention are deliveredwith specificity for human tumor cells, specifically non-apoptotic humantumor cells including for example T-cells, hepatocytes, breast carcinomacells, ovarian carcinoma cells, melanoma cells, intestinal epithelialcells, prostate cells, testicular cells, non-small cell lung cancers,small cell lung cancers, etc.

Alternatively, certain siNA molecules of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.Natl. Acad. Sci., USA 83, 399; Thompson et al., 1995, Nucleic AcidsRes., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45; Noonberg et al.,5,624,803; Thompson, U.S. Pat. Nos. 5,902,880 and 6,146,886; Paul etal., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002,Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology,19, 500; for a review see Couture et al., 1996, TIG., 12, 510). Thoseskilled in the art realize that any nucleic acid can be expressed ineukaryotic cells from the appropriate DNA/RNA vector. The activity ofsuch nucleic acids can be augmented by their release from the primarytranscript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569,and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic AcidsSymp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19,5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowriraet al., 1994, J. Biol. Chem., 269, 25856).

In another aspect of the invention, siNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Thompson, U.S. Pat.Nos. 5,902,880 and 6,146,886). The recombinant vectors capable ofexpressing the siNA molecules can be delivered as described above, andpersist in target cells. Alternatively, viral vectors can be used thatprovide for transient expression of nucleic acid molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecule interacts with the target mRNA and generates an RNAi response.Delivery of siNA molecule expressing vectors can be systemic, such as byintravenous or intra-muscular administration, by administration totarget cells ex-planted from a subject followed by reintroduction intothe subject, or by any other means that would allow for introductioninto the desired target cell (for a review see Couture et al., 1996,TIG., 12, 510).

EXAMPLES

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1 Serum Stability of Chemically Modified siNA Constructs

Chemical modifications are introduced into siNA constructs to determinethe stability of these constructs compared to native siNAoligonucleotides (or those containing for example two thymidinenucleotide overhangs) in human serum. RNAi stability tests are performedby internally labeling siNA and duplexing by incubating in appropriatebuffers to facilitate the formation of duplexes by the siNA. DuplexedsiNA constructs are then tested for stability by incubating at a finalconcentration of 2 μM siNA (strand 2 concentration) in 90% mouse orhuman serum for time-points of 30 sec, 1 min, 5 min, 30 min, 90 min, 4hrs 10 min, 16 hrs 24 min, and 49 hrs. Time points are run on a 15%denaturing polyacrylamide gels and analyzed on a phosphoimager.

Internal labeling is performed via kinase reactions with polynucleotidekinase (PNK) and ³²P-γ-ATP, with addition of radiolabeled phosphate at anucleotide position (e.g. nucleotide 13) of strand 2, counting in fromthe 3′ side. Ligation of the remaining fragments with T4 RNA ligaseresults in the full length strand 2. Duplexing of siNA is accomplishedfor example by adding an appropriate concentration of the siNAoligonucleotide and heating to 95° C. for 5 minutes followed by slowcooling to room temperature. Reactions are performed by adding 100%serum to the siNA duplexes and incubating at 37° C., then removingaliquots at desired time-points.

Example 2 Identification of Potential siNA Target Sites in any RNASequence

The sequence of an RNA target of interest, such as a viral or human mRNAtranscript, is screened for target sites, for example by using acomputer folding algorithm. Such target sites can contain complementary,palindrome or repeat sequences that are shared by more than one targetnucleic acid sequence such that multifunctional siNA molecules can bedesigned to target differing nucleic acid sequences sharing commonpalindrome or repeat sequences or having some degree of selfcomplementarity. In addition, the use of non-naturally occurringnucleotides or non-nucleotides can be utilized to generate artificialcomplementary, palindrome, or repeat regions within a multifunctionalsiNA molecule of the invention (see for example FIG. 7). In anon-limiting example, the sequence of a gene or RNA gene transcriptderived from a database, such as Genbank, is used to generate siNAsequences having complementarity to the target. Such sequences can beobtained from a database, or can be determined experimentally as knownin the art. Target sites that are known, for example, those target sitesdetermined to be effective target sites based on studies with othernucleic acid molecules, for example ribozymes or antisense, or thosetargets known to be associated with a disease or condition such as thosesites containing mutations or deletions, can be used to design siNAmolecules targeting those sites. Various parameters can be used todetermine which sites are the most suitable target sites within thetarget RNA sequence. These parameters include but are not limited tosecondary or tertiary RNA structure, the nucleotide base composition ofthe target sequence, the degree of homology between various regions ofthe target sequence, or the relative position of the target sequencewithin the RNA transcript. Based on these determinations, any number oftarget sites within the RNA transcript can be chosen to screen siNAmolecules for efficacy, for example by using in vitro RNA cleavageassays, cell culture, or animal models. In a non-limiting example,anywhere from 1 to 1000 target sites are chosen within the transcriptbased on the size of the siNA construct to be used. High throughputscreening assays can be developed for screening siNA molecules usingmethods known in the art, such as with multi-well or multi-plate assaysor combinatorial/siNA library screening assays to determine efficientreduction in target gene expression.

In a non-limiting example, a multifunctional siNA is designed to targettwo separate nucleic acid sequences. The goal is to combine twodifferent siNAs together in one siNA that is active against twodifferent targets. The siNAs are joined in a way that the 5′ of eachstrand starts with the “antisense” sequence of one of two siRNAs asshown in italics below.

SEQ ID NO: 1 3′TTAGAAACCAGACGUAAGUGU GGUACGACCUGACGACCGU 5′ SEQ ID NO: 25′ UCUUUGGUCUGCAUUCACAC CAUGCUGGACUGCUGGCATT3′

RISC is expected to incorporate either of the two strands from 5′ end.This would lead to two types of active RISC populations carrying eitherstrand. The 5′ 19 nt of each strand will act as guide sequence fordegradation of separate target sequences.

In another example, the size of multifunctional siNA molecules isreduced by either finding overlaps or truncating the individual siNAlength. The exemplary exercise described below indicates that for anygiven first target sequence, a shared complementary sequence in a secondtarget sequence is likely to be found.

The number of spontaneous matches of short polynucleotide sequences(e.g., less than 14 nucleotides) that are expected to occur between twolonger sequences generated independent of one another was investigated.A simulation using the uniform random generator SAS V8.1 utilized a4,000 character string that was generated as a random repeatingoccurrence of the letters {ACGU}. This sequence was then broken into thenearly 4000 overlapping sets formed by taking S1 as the characters frompositions (1, 2 . . . n), S2 from positions (2,3 . . . , n+1) completelythrough the sequence to the last set, S 4000-n+1 from position(4000-n+1, . . . ,4000). This process was then repeated for a second4000 character string. Occurrence of same sets (of size n) were thenchecked for sequence identity between the two strings by a sorting andmatch-merging routine. This procedure was repeated for sets of 9-11characters. Results were an average of 55 matching sequences of lengthn=9 characters (range 39 to 72); 13 common sets (range 6 to 18) for sizen=10, and 4 matches on average (range 0 to 6) for sets of 11 characters.The choice of 4000 for the original string length is approximately thelength of the coding region of both VEGFR1 and VEGFR2. This simplesimulation suggests that any two long coding regions formed independentof one-another will share common short sequences that can be used toshorten the length of multifunctional siNA constructs. In this example,common sequences of size 9 occurred by chance alone in >1% frequency.

Below is an example of a multifunctional siNA construct that targetsVEGFR1 and VEGFR2 in which each strand has a total length of 24 nt witha 14 nt self complementary region (underline). The antisense region ofeach siNA ‘1’ targeting VEGFR1 and siNA ‘2’ targeting VEGFR2(complementary regions are shown in italic) are used

siNA ‘1’ 5′ CAAUUAGAGUGGCAGUGAG (SEQ ID NO: 3) 3′ GUUAAUCUCACCGUCACUC(SEQ ID NO: 4) siNA ‘2’ AGAGUGGCAGUGAGCAAAG 5′ (SEQ ID NO: 5)UCUCACCGUCACUCGUUUC 3′ (SEQ ID NO: 6) Multifunctional siNA CAAUUAGAGUGGCAGUGAG CAAAG (SEQ ID NO: 7) GUUAA UCUCACCGUCACUC GUUUC(SEQ ID NO: 8)

In another example, the length of a multifunctional siNA construct isreduced by determining whether fewer base pairs of sequence homology toeach target sequence can be tolerated for effective RNAi activity. Ifso, the overall length of multifunctional siNA can be reduced as shownbelow. In the following hypothetical example, 4 nucleotides (bold) arereduced from each 19 nucleotide siNA ‘1’ and siNA ‘2’ constructs. Theresulting multifunctional siNA is 30 base pairs long.

siNA ‘1’ 5′ CAAUUAGAGUGGCAG

(SEQ ID NO: 3) 3′ GUUAAUCUCACCGUCACUC (SEQ ID NO: 4) siNA ‘2’AGAGUGGCAGUGAGCAAAG 5′ (SEQ ID NO: 5)

ACCGUCACUCGUUUC 3′ (SEQ ID NO: 6) Multifunctional siNACAAUUAGAGUGGCAGUGGCAGUGAGCAAAG (SEQ ID NO: 9)GUUAAUCUCACCGUCACCGUCACUCGUUUC (SEQ ID NO: 10)

Example 3 Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

The target sequence is parsed in silico into a list of all fragments orsubsequences of a particular length, for example 23 nucleotidefragments, contained within the target sequence. This step is typicallycarried out using a custom Perl script, but commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package can beemployed as well.

In some instances, the siNAs correspond to more than one targetsequence; such would be the case for example in targeting differenttranscripts of the same gene, targeting different transcripts of morethan one gene, or for targeting both the human gene and an animalhomolog. In this case, a subsequence list of a particular length isgenerated for each of the targets, and then the lists are compared tofind matching sequences in each list. The subsequences are then rankedaccording to the number of target sequences that contain the givensubsequence. The goal is to find subsequences that are present in mostor all of the target sequences. Alternately, the ranking can identifysubsequences that are unique to a target sequence, such as a mutanttarget sequence. Such an approach would enable the use of siNA to targetspecifically the mutant sequence and not effect the expression of thenormal sequence.

In some instances, the siNA subsequences are absent in one or moresequences while present in the desired target sequence; such would bethe case if the siNA targets a gene with a paralogous family member thatis to remain untargeted. As in case 2 above, a subsequence list of aparticular length is generated for each of the targets, and then thelists are compared to find sequences that are present in the target genebut are absent in the untargeted paralog.

The ranked siNA subsequences can be further analyzed and rankedaccording to GC content. A preference can be given to sites containing30-70% GC, with a further preference to sites containing 40-60% GC.

The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have runs of GGG or CCC in the sequence. GGG(or even more Gs) in either strand can make oligonucleotide synthesisproblematic and can potentially interfere with activity, so it isavoided when other appropriately suitable sequences are available. CCCis searched in the target strand because that will place GGG in the siNAstrand.

The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have the dinucleotide UU (uridinedinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end ofthe sequence (to yield 3′ UU on the siNA sequence). These sequencesallow one to design siNA molecules with terminal TT thymidinedinucleotides.

Other design considerations can be used when selecting target nucleicacid sequences, see for example Reynolds et al., 2004, NatureBiotechnology Advanced Online Publication, 1 Feb. 2004,doi:10.1038/nbt936.

The siNA molecules are screened in an appropriate in vitro, cell cultureor animal model system, such as the systems described herein orotherwise known in the art, to identify the most active siNA molecule orthe most preferred target sites within the target RNA sequences.

Example 4 siNA Design

siNA target sites were chosen by analyzing sequences of the target RNAand optionally prioritizing the target sites on the basis of preferredsequence motifs, such as predicted duplex stability, GC content, folding(structure of any given sequence analyzed to determine siNAaccessibility to the target), other parameters as are known in the art(see for example Reynolds et al., 2004, Nature Biotechnology AdvancedOnline Publication, 1 Feb. 2004, doi:10.1038/nbt936), or by using alibrary of siNA molecules.

Once target sites have been identified for multifunctional siNAconstructs, each strand of the siNA is designed with a complementaryregion of length, for example, between about 18 and about 28nucleotides, that is complementary to a different target nucleic acidsequence. Each complementary region is designed with an adjacentflanking region of about 4 to about 22 nucleotides that is notcomplementary to the target sequence, but which comprisescomplementarity to the complementary region of the other sequence (seefor example FIG. 1). Hairpin constructs can likewise be designed (seefor example FIG. 2). Identification of complementary, palindrome orrepeat sequences that are shared between the different target nucleicacid sequences can be used to shorten the overall length of themultifunctional siNA constructs (see for example FIGS. 3 and 4).

siNA molecules are designed that could bind each target and areoptionally individually analyzed by computer folding to assess whetherthe siNA molecule can interact with the target sequence. Varying thelength of the siNA molecules can be chosen to optimize activity.Generally, a sufficient number of complementary nucleotide bases arechosen to bind to, or otherwise interact with, the target RNA sequences,but the degree of complementarity can be modulated to accommodate siNAduplexes or varying length or base composition. By using suchmethodologies, siNA molecules can be designed to target sites within anycombination of known RNA sequences, for example those RNA sequencescorresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nucleasestability for systemic administration in vivo and/or improvedpharmacokinetic, localization, and delivery properties while preservingthe ability to mediate gene inhibition activity. Chemical modificationsas described herein are introduced synthetically using synthetic methodsdescribed herein and those generally known in the art. The syntheticsiNA constructs are then assayed for nuclease stability in serum and/orcellular/tissue extracts (e.g. liver extracts). The synthetic siNAconstructs are also tested in parallel for activity using an appropriateassay, such as a luciferase reporter assay as described herein oranother suitable assay that can quantity inhibitory activity. SyntheticsiNA constructs that possess both nuclease stability and activity can befurther modified and re-evaluated in stability and activity assays. Thechemical modifications of the stabilized active siNA constructs can thenbe applied to any siNA sequence targeting any chosen RNA and used, forexample, in target screening assays to pick lead siNA compounds fortherapeutic development. Alternately, chemically modified siNAconstructs can be screened directly for activity in an appropriate assaysystem (e.g., cell culture, animal models etc.).

Example 5 Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage (see for example FIG. 6), for example, target sequences withinthe RNA sequences described herein or with various sites in differentRNA sequences (see for example FIG. 5). The sequence of the siNAmolecule(s) is complementary to the target site sequences describedabove. The siNA molecules can be chemically synthesized using methodsdescribed herein. Inactive siNA molecules that are used as controlsequences can be synthesized by scrambling the sequence of the siNAmolecules such that it is not complementary to the target sequence.Generally, siNA constructs can by synthesized using solid phaseoligonucleotide synthesis methods as described herein (see for exampleUsman et al., U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657;6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat.Nos. 6,111,086; 6,008,400; 6,111,086 all incorporated by referenceherein in their entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry described hereinand as is known in the art. Standard phosphoramidite chemistry involvesthe use of nucleosides comprising any of 5′-O-dimethoxytrityl,2′-O-tert-butyldimethylsilyl, 3′-O-2-CyanoethylN,N-diisopropylphos-phoroamidite groups, and exocyclic amine protectinggroups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyrylguanosine). Alternately, 2′-O—Silyl ethers can be used in conjunctionwith acid-labile 2′-O-orthoester protecting groups in the synthesis ofRNA as described by Scaringe supra. Differing 2′ chemistries can requiredifferent protecting groups, for example, 2′-deoxy-2′-amino nucleosidescan utilize N-phthaloyl protection as described by Usman et al., U.S.Pat. No. 5,631,360, incorporated by reference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and Fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example, by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat.No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No.6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringesupra, incorporated by reference herein in their entireties.Additionally, deprotection conditions can be modified to provide thebest possible yield and purity of siNA constructs. For example,applicant has observed that oligonucleotides comprising2′-deoxy-2′-fluoro nucleotides can degrade under inappropriatedeprotection conditions. Such oligonucleotides are deprotected usingaqueous methylamine at about 35° C. for 30 minutes. If the2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 6 Nucleic Acid Inhibition of Target RNA In Vivo

siNA molecules targeted to the target RNA are designed and synthesizedas described above. These nucleic acid molecules can be tested forcleavage activity in vivo, for example, using the following procedure.

Two formats are used to test the efficacy of siNAs targeting aparticular gene transcript. First, the reagents are tested on targetexpressing cells (e.g., HeLa), to determine the extent of RNA andprotein inhibition. siNA reagents are selected against the RNA target.RNA inhibition is measured after delivery of these reagents by asuitable transfection agent to cells. Relative amounts of target RNA aremeasured versus actin using real-time PCR monitoring of amplification(e.g., ABI 7700 Taqman®). A comparison is made to a mixture ofoligonucleotide sequences made to unrelated targets or to a randomizedsiNA control with the same overall length and chemistry, but withrandomly substituted nucleotides at each position. Primary and secondarylead reagents are chosen for the target and optimization performed.After an optimal transfection agent concentration is chosen, a RNAtime-course of inhibition is performed with the lead siNA molecule. Inaddition, a cell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells

Cells (e.g., HeLa) are seeded, for example, at 1×10⁵ cells per well of asix-well dish in EGM-2 (BioWhittaker) the day before transfection. siNA(final concentration, for example 20 nM) and cationic lipid (e.g., finalconcentration 2 μg/ml) are complexed in EGM basal media (Biowhittaker)at 37° C. for 30 mins in polystyrene tubes. Following vortexing, thecomplexed siNA is added to each well and incubated for the timesindicated. For initial optimization experiments, cells are seeded, forexample, at 1×10³ in 96 well plates and siNA complex added as described.Efficiency of delivery of siNA to cells is determined using afluorescent siNA complexed with lipid. Cells in 6-well dishes areincubated with siNA for 24 hours, rinsed with PBS and fixed in 2%paraformaldehyde for 15 minutes at room temperature. Uptake of siNA isvisualized using a fluorescent microscope.

Taqman and LightCycler Quantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For Taqman analysis, dual-labeled probes aresynthesized with the reporter dye, FAM or JOE, covalently linked at the5′-end and the quencher dye TAMRA conjugated to the 3′-end. One-stepRT-PCR amplifications are performed on, for example, an ABI PRISM 7700Sequence Detector using 50 μl reactions consisting of 10 μl total RNA,100 nM forward primer, 900 nM reverse primer, 100 nM probe, 1× TaqManPCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM eachdATP, dCTP, dGTP, and dTTP, 10 U RNase Inhibitor (Promega), 1.25 UAmpliTaq Gold (PE-Applied Biosystems) and 10 U M-MLV ReverseTranscriptase (Promega). The thermal cycling conditions can consist of30 min at 48° C., 10 min at 95° C., followed by 40 cycles of 15 sec at95° C. and 1 min at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/rxn) and normalizing to β-actin or GAPDH mRNA inparallel TaqMan reactions. For each gene of interest an upper and lowerprimer and a fluorescently labeled probe are designed. Real timeincorporation of SYBR Green I dye into a specific PCR product can bemeasured in glass capillary tubes using a lightcyler. A standard curveis generated for each primer pair using control cRNA. Values arerepresented as relative expression to GAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example, using TCA precipitation. An equal volume of 20% TCA isadded to the cell supernatant, incubated on ice for 1 hour and pelletedby centrifugation for 5 minutes. Pellets are washed in acetone, driedand resuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example, (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 7 Multifunctional siNA Constructs Targeting VEGF and VEGFReceptors

Using the methods described herein, multifunctional siNA constructs aredesigned against VEGF and VEGFR (e.g. VEGFR1 and/or VEGFR2) target RNAsequences. These siNA constructs can utilize tandem sequences of bothtargets that do not share any complementarity (see for example FIGS. 1and 2). Alternately, the siNA constructs can utilize the identificationof complementary, palindromic or repeat sequences (for example Z inFormula I herein) in target nucleic acid sequences of interest (see forexample FIGS. 3 and 4). Generally these complementary palindrome/repeatsequences comprise about 2 to about 12 nucleotides in length, but canvary according to a particular multifunctional siNA construct. In oneexample, a nucleotide sequence that is complementary to the VEGF targetnucleic acid sequence is incorporated at the 3′-end of the first strandof the siNA molecule and a nucleotide sequence that is complementary tothe VEGFR target nucleic acid sequence is incorporated at the 3′-end ofthe second strand of the siNA molecule (e.g., FIG. 1A). Alternately, thenucleotide sequence that is complementary to the VEGF target nucleicacid sequence is incorporated at the 5′-end of the first strand of thesiNA molecule and a nucleotide sequence that is complementary to theVEGFR target nucleic acid sequence is incorporated at the 5′-end of thesecond strand of the siNA molecule (e.g., FIG. 1B). If selfcomplementary, palindrome or repeat sequences are used, then generally,the longer the repeat identified in the target nucleic acid sequence,the shorter the resulting siNA sequence will be. For example, if eachtarget sequence is 21 nucleotides in length and there is no repeat foundin the sequence, the resulting siNA construct will be, for example,21+0+21=42 nucleotides in length. The first 21 nucleotides representsequence complementary to the first target nucleic acid sequence, the 0represents the lack of a self complementary, palindrome, or repeatsequence, and the second 21 nucleotides represent sequence complementaryto the second target nucleic acid sequence. If a 2 nucleotide repeat isutilized, the resulting siNA construct will be, for example, 19+2+19=40nucleotides in length. If a 4 nucleotide repeat is utilized, theresulting siNA construct will be, for example, 17+4+17=38 nucleotides inlength. If a 6 nucleotide repeat is utilized, the resulting siNAconstruct will be, for example, 15+6+15=36 nucleotides in length. If an8 nucleotide repeat is utilized, the resulting siNA construct will be,for example, 13+8+13=34 nucleotides in length. If a 10 nucleotide repeatis utilized, the resulting siNA construct will be, for example,11+10+11=32 nucleotides in length. If a 12 nucleotide repeat isutilized, the resulting siNA construct will be, for example, 9+12+9=30nucleotides in length and so forth. Thus, for each nucleotide reductionin the target site, the siNA length can be shortened by 2 nucleotides.These same principles can be utilized for a target site having differentlength nucleotide sequences, such as target sites comprising 14 to 28nucleotides. In addition, various combinations of 5′ and 3′ overhangsequences (e.g., TT) can be introduced to the siNA constructs designedwith self complementary, palindrome, or repeat sequences.

In one example, multifunctional siNA are designed against VEGFR1 andVEGFR2 RNA targets and are screened in cell culture experiments alongwith chemically modified siNA constructs with known activity withmatched chemistry inverted controls and untreated cells along with atrasfection control (LF2K). In a non-limiting example, multifunctionalsiNA sequences targeting VEGFR1 and VEGFR2 comprise sequences shown inTable I. HAEC cells are transfected with 0.25 ug/well of lipid complexedwith 25 nM multifunctional siNA targeting for example VEGFR1 site 1501and VEGFR2 site 5760. Cells are incubated at 37° for 24 h in thecontinued presence of the siNA transfection mixture. At 24 h, RNA isprepared from each well of treated cells. The supernatants with thetransfection mixtures are first removed and discarded, then the cellsare lysed and RNA prepared from each well. Target gene expressionfollowing treatment is evaluated by RT-PCR for the VEGFR1 and VEGFR2mRNA and for a control gene (36B4, an RNA polymerase subunit) fornormalization.

In one example, multifunctional siNA are designed against VEGF andVEGFR1RNA targets and are screened in cell culture experiments alongwith chemically modified siNA constructs with known activity withmatched chemistry inverted controls and untreated cells along with atrasfection control (LF2K). In a non-limiting example, multifunctionalsiNA sequences targeting VEGF and VEGFR1 comprise sequences shown inTable II. HAEC cells are transfected with 0.25 ug/well of lipidcomplexed with 25 nM multifunctional siNA targeting for example VEGFR1site 5353 and VEGF site 360. Cells are incubated at 37° for 24 h in thecontinued presence of the siNA transfection mixture. At 24 h, RNA isprepared from each well of treated cells. The supernatants with thetransfection mixtures are first removed and discarded, then the cellsare lysed and RNA prepared from each well. Target gene expressionfollowing treatment is evaluated by RT-PCR for the VEGFR1 and VEGF mRNAand for a control gene (36B4, an RNA polymerase subunit) fornormalization.

In one example, multifunctional siNA are designed against VEGF andVEGFR2RNA targets and are screened in cell culture experiments alongwith chemically modified siNA constructs with known activity withmatched chemistry inverted controls and untreated cells along with atrasfection control (LF2K). In a non-limiting example, multifunctionalsiNA sequences targeting VEGF and VEGFR2 comprise sequences shown inTable III. HAEC cells are transfected with 0.25 ug/well of lipidcomplexed with 25 nM multifunctional siNA targeting for example VEGFR2site 905 and VEGF site 220. Cells are incubated at 37° for 24 h in thecontinued presence of the siNA transfection mixture. At 24 h, RNA isprepared from each well of treated cells. The supernatants with thetransfection mixtures are first removed and discarded, then the cellsare lysed and RNA prepared from each well. Target gene expressionfollowing treatment is evaluated by RT-PCR for the VEGFR2 and VEGF mRNAand for a control gene (36B4, an RNA polymerase subunit) fornormalization.

In one example, multifunctional siNA are designed against VEGF RNAtargets and conserved sites within VEGFR1/VEGFR2RNA targets and arescreened in cell culture experiments along with chemically modified siNAconstructs with known activity with matched chemistry inverted controlsand untreated cells along with a trasfection control (LF2K). In anon-limiting example, multifunctional siNA sequences targeting VEGF,VEGFR1, and VEGFR2 comprise sequences shown in Table IV. HAEC cells aretransfected with 0.25 ug/well of lipid complexed with 25 nMmultifunctional siNA targeting for example VEGFR1/R2 hybrid site 3646and VEGF site 349. Cells are incubated at 37° for 24 h in the continuedpresence of the siNA transfection mixture. At 24 h, RNA is prepared fromeach well of treated cells. The supernatants with the transfectionmixtures are first removed and discarded, then the cells are lysed andRNA prepared from each well. Target gene expression following treatmentis evaluated by RT-PCR for the VEGFR1, VEGFR2 and VEGF mRNA and for acontrol gene (36B4, an RNA polymerase subunit) for normalization.

Example 8 Multifunctional siNA Constructs Targeting HBV and FAS RNA

Using the methods described herein, multifunctional siNA constructs aredesigned against HBV and FAS target RNA sequences. These siNA constructscan utilize tandem sequences of both targets that do not share anycomplementarity (see for example FIGS. 1 and 2). Alternately, the siNAconstructs can utilize the identification of complementary, palindromicor repeat sequences (for example Z in Formula I herein) in targetnucleic acid sequences of interest (see for example FIGS. 3 and 4).Generally these complementary palindrome/repeat sequences comprise about2 to about 12 nucleotides in length, but can vary according to aparticular multifunctional siNA construct. In one example, a nucleotidesequence that is complementary to the HBV target nucleic acid sequenceis incorporated at the 3′-end of the first strand of the siNA moleculeand a nucleotide sequence that is complementary to the FAS targetnucleic acid sequence is incorporated at the 3′-end of the second strandof the siNA molecule (e.g., FIG. 1A). Alternately, the nucleotidesequence that is complementary to the HBV target nucleic acid sequenceis incorporated at the 5′-end of the first strand of the siNA moleculeand a nucleotide sequence that is complementary to the FAS receptortarget nucleic acid sequence is incorporated at the 5′-end of the secondstrand of the siNA molecule (e.g., FIG. 1B). If self complementary,palindrome or repeat sequences are used, then generally, the longer therepeat identified in the target nucleic acid sequence, the shorter theresulting siNA sequence will be. For example, if each target sequence is21 nucleotides in length and there is no repeat found in the sequence,the resulting siNA construct will be, for example, 21+0+21=42nucleotides in length. The first 21 nucleotides represent sequencecomplementary to the first target nucleic acid sequence, the 0represents the lack of a self complementary, palindrome, or repeatsequence, and the second 21 nucleotides represent sequence complementaryto the second target nucleic acid sequence. If a 2 nucleotide repeat isutilized, the resulting siNA construct will be, for example, 19+2+19=40nucleotides in length. If a 4 nucleotide repeat is utilized, theresulting siNA construct will be, for example, 17+4+17=38 nucleotides inlength. If a 6 nucleotide repeat is utilized, the resulting siNAconstruct will be, for example, 15+6+15=36 nucleotides in length. If an8 nucleotide repeat is utilized, the resulting siNA construct will be,for example, 13+8+13=34 nucleotides in length. If a 10 nucleotide repeatis utilized, the resulting siNA construct will be, for example,11+10+11=32 nucleotides in length. If a 12 nucleotide repeat isutilized, the resulting siNA construct will be, for example, 9+12+9=30nucleotides in length and so forth. Thus, for each nucleotide reductionin the target site, the siNA length can be shortened by 2 nucleotides.These same principles can be utilized for a target site having differentlength nucleotide sequences, such as target sites comprising 14 to 28nucleotides. In addition, various combinations of 5′ and 3′ overhangsequences (e.g., TT) can be introduced to the siNA constructs designedwith self complementary, palindrome, or repeat sequences.

In one example, multifunctional siNA are designed against HBV and FASRNA targets as described herein and are screened in HepG2 cells.Transfection of the human hepatocellular carcinoma cell line, Hep G2,with replication-competent HBV DNA results in the expression of HBVproteins and the production of virions. The human hepatocellularcarcinoma cell line Hep G2 is grown in Dulbecco's modified Eagle mediasupplemented with 10% fetal calf serum, 2 mM glutamine, 0.1 mMnonessential amino acids, 1 mM sodium pyruvate, 25 mM Hepes, 100 unitspenicillin, and 100 μg/ml streptomycin. To generate a replicationcompetent cDNA, prior to transfection the HBV genomic sequences areexcised from the bacterial plasmid sequence contained in the psHBV-1vector. Other methods known in the art can be used to generate areplication competent cDNA. This can be done with an EcoRI and Hind IIIrestriction digest. Following completion of the digest, a ligation isperformed under dilute conditions (20 μg/ml) to favor intermolecularligation. The total ligation mixture is then concentrated using Qiagenspin columns. To test the efficacy of siNAs targeted against both HBVand FAS RNA, multifunctional siNA duplexes targeting HBV pregenomic RNAand FAS RNA are co-transfected with HBV genomic DNA once at 25 nM withlipid at 12.5 ug/ml into Hep G2 cells, and the subsequent levels ofsecreted HBV surface antigen (HBsAg) is analyzed by ELISA and FAS RNA isquantitated by RT-PCR.

Example 9 Multifunctional siNA Constructs Targeting HCV and FAS RNA

Using the methods described herein, multifunctional siNA constructs aredesigned against HCV and FAS target RNA sequences. These siNA constructscan utilize tandem sequences of both targets that do not share anycomplementarity (see for example FIGS. 1 and 2). Alternately, the siNAconstructs can utilize the identification of complementary, palindromicor repeat sequences (for example Z in Formula I herein) in targetnucleic acid sequences of interest (see for example FIGS. 3 and 4).Generally these complementary palindrome/repeat sequences comprise about2 to about 12 nucleotides in length, but can vary according to aparticular multifunctional siNA construct. In one example, a nucleotidesequence that is complementary to the HCV target nucleic acid sequenceis incorporated at the 3′-end of the first strand of the siNA moleculeand a nucleotide sequence that is complementary to the FAS targetnucleic acid sequence is incorporated at the 3′-end of the second strandof the siNA molecule (e.g., FIG. 1A). Alternately, the nucleotidesequence that is complementary to the HCV target nucleic acid sequenceis incorporated at the 5′-end of the first strand of the siNA moleculeand a nucleotide sequence that is complementary to the FAS receptortarget nucleic acid sequence is incorporated at the 5′-end of the secondstrand of the siNA molecule (e.g., FIG. 1B). If self complementary,palindrome or repeat sequences are used, then generally, the longer therepeat identified in the target nucleic acid sequence, the shorter theresulting siNA sequence will be. For example, if each target sequence is21 nucleotides in length and there is no repeat found in the sequence,the resulting siNA construct will be, for example, 21+0+21=42nucleotides in length. The first 21 nucleotides represent sequencecomplementary to the first target nucleic acid sequence, the 0represents the lack of a self complementary, palindrome, or repeatsequence, and the second 21 nucleotides represent sequence complementaryto the second target nucleic acid sequence. If a 2 nucleotide repeat isutilized, the resulting siNA construct will be, for example, 19+2+19=40nucleotides in length. If a 4 nucleotide repeat is utilized, theresulting siNA construct will be, for example, 17+4+17=38 nucleotides inlength. If a 6 nucleotide repeat is utilized, the resulting siNAconstruct will be, for example, 15+6+15=36 nucleotides in length. If an8 nucleotide repeat is utilized, the resulting siNA construct will be,for example, 13+8+13=34 nucleotides in length. If a 10 nucleotide repeatis utilized, the resulting siNA construct will be, for example,11+10+11=32 nucleotides in length. If a 12 nucleotide repeat isutilized, the resulting siNA construct will be, for example, 9+12+9=30nucleotides in length and so forth. Thus, for each nucleotide reductionin the target site, the siNA length can be shortened by 2 nucleotides.These same principles can be utilized for a target site having differentlength nucleotide sequences, such as target sites comprising 14 to 28nucleotides. In addition, various combinations of 5′ and 3′ overhangsequences (e.g., TT) can be introduced to the siNA constructs designedwith self complementary, palindrome, or repeat sequences.

In one example, a HCV replicon system is used to test the efficacy ofsiNAs targeting HCV target RNA and FAS target RNA. The reagents aretested in cell culture using Huh7 cells (see for example Randall et al.,2003, PNAS USA, 100, 235-240) to determine the extent of HCV and FAS RNAand/or protein inhibition. Multifunctional siNAs are selected againstthe HCV and FAS nucleic acid targets as described herein. In anon-limiting example, multifunctional siNA sequences targeting HCV RNAand FAS RNA comprise sequences shown in Table V. RNA inhibition ismeasured after delivery of these reagents by a suitable transfectionagent to Huh7 cells. Relative amounts of target RNA are measured versusactin using real-time PCR monitoring of amplification (e.g., ABI 7700Taqman®). A comparison is made to a mixture of oligonucleotide sequencesdesigned to target unrelated targets or to a randomized siNA controlwith the same overall length and chemistry, but with randomlysubstituted nucleotides at each position. Primary and secondary leadreagents are chosen for the target and optimization performed. After anoptimal transfection agent concentration is chosen, a RNA time-course ofinhibition is performed with the lead multifunctional siNA molecule. Inaddition, a cell-plating format can be used to determine RNA inhibition.Generally, siNA reagents are transfected at 25 nM into Huh7 cells andHCV RNA and FAS RNA is quantitated compared to untreated cells and cellstransfected with lipofectamine (“LFA2K) as a transfection control.

Example 10 Multifunctional siNA Constructs Targeting TGF-Beta andTGF-Beta Receptor RNA

Using the methods described herein, multifunctional siNA constructs aredesigned against TGF-beta and TGF-beta receptor target RNA sequences.These siNA constructs can utilize tandem sequences of both targets thatdo not share any complementarity (see for example FIGS. 1 and 2).Alternately, the siNA constructs can utilize the identification ofcomplementary, palindromic or repeat sequences (for example Z in FormulaI herein) in target nucleic acid sequences of interest (see for exampleFIGS. 3 and 4). Generally these complementary palindrome/repeatsequences comprise about 2 to about 12 nucleotides in length, but canvary according to a particular multifunctional siNA construct. In oneexample, a nucleotide sequence that is complementary to the TGF-betatarget nucleic acid sequence is incorporated at the 3′-end of the firststrand of the siNA molecule and a nucleotide sequence that iscomplementary to the TGF-beta receptor target nucleic acid sequence isincorporated at the 3′-end of the second strand of the siNA molecule(e.g., FIG. 1A). Alternately, the nucleotide sequence that iscomplementary to the TGF-beta target nucleic acid sequence isincorporated at the 5′-end of the first strand of the siNA molecule anda nucleotide sequence that is complementary to the TGF-beta receptortarget nucleic acid sequence is incorporated at the 5′-end of the secondstrand of the siNA molecule (e.g., FIG. 1B). If self complementary,palindrome or repeat sequences are used, then generally, the longer therepeat identified in the target nucleic acid sequence, the shorter theresulting siNA sequence will be. For example, if each target sequence is21 nucleotides in length and there is no repeat found in the sequence,the resulting siNA construct will be, for example, 21+0+21=42nucleotides in length. The first 21 nucleotides represent sequencecomplementary to the first target nucleic acid sequence, the 0represents the lack of a self complementary, palindrome, or repeatsequence, and the second 21 nucleotides represent sequence complementaryto the second target nucleic acid sequence. If a 2 nucleotide repeat isutilized, the resulting siNA construct will be, for example, 19+2+19=40nucleotides in length. If a 4 nucleotide repeat is utilized, theresulting siNA construct will be, for example, 17+4+17=38 nucleotides inlength. If a 6 nucleotide repeat is utilized, the resulting siNAconstruct will be, for example, 15+6+15=36 nucleotides in length. If an8 nucleotide repeat is utilized, the resulting siNA construct will be,for example, 13+8+13=34 nucleotides in length. If a 10 nucleotide repeatis utilized, the resulting siNA construct will be, for example,11+10+11=32 nucleotides in length. If a 12 nucleotide repeat isutilized, the resulting siNA construct will be, for example, 9+12+9=30nucleotides in length and so forth. Thus, for each nucleotide reductionin the target site, the siNA length can be shortened by 2 nucleotides.These same principles can be utilized for a target site having differentlength nucleotide sequences, such as target sites comprising 14 to 28nucleotides. In addition, various combinations of 5′ and 3′ overhangsequences (e.g., TT) can be introduced to the siNA constructs designedwith self complementary, palindrome, or repeat sequences.

In one example, multifunctional siNA are designed against TGF-beta andTGF-beta receptor targets and are screened in cell culture experimentsalong with chemically modified siNA constructs with known activity usingmatched chemistry inverted controls and untreated cells along with atrasfection control (LF2K). In a non-limiting example, multifunctionalsiNA sequences targeting TGF-beta and TGF-beta receptor comprisesequences shown in Table VI. A549 cells are transfected with 0.25ug/well of lipid complexed with 25 nM multifunctional siNA targeting forexample TGF-beta site 169 and TGF-beta receptor site 127. Cells areincubated at 37° for 24 h in the continued presence of the siNAtransfection mixture. At 24 h, RNA is prepared from each well of treatedcells. The supernatants with the transfection mixtures are first removedand discarded, then the cells are lysed and RNA prepared from each well.Target gene expression following treatment is evaluated by RT-PCR forthe TGF-beta and TGF-beta receptor mRNA and for a control gene (36B4, anRNA polymerase subunit) for normalization.

Example 11 Multifunctional siNA Constructs Targeting HIV and CellularRNA

Using the methods described herein, multifunctional siNA constructs aredesigned against HIV and cellular target RNA sequences. Non-limitingexamples of HIV targets include HIV LTR, HIV-TAT, HIV-REV, HIV-NEF,HIV-RRE, HIV-TAR, HIV-VIF, and HIV-ENF. Non-limiting examples ofcellular targets include CD4 receptors, CXCR4, CCR5, CCR3, CCR2, CCR1,CCR4, CCR8, CCR9, CXCR2, STRL33 and others described herein. These siNAconstructs can utilize tandem sequences of both targets that do notshare any complementarity (see for example FIGS. 1 and 2). Alternately,the siNA constructs can utilize the identification of complementary,palindromic or repeat sequences (for example Z in Formula I herein) intarget nucleic acid sequences of interest (see for example FIGS. 3 and4). Generally these complementary palindrome/repeat sequences compriseabout 2 to about 12 nucleotides in length, but can vary according to aparticular multifunctional siNA construct. In one example, a nucleotidesequence that is complementary to the HIV target nucleic acid sequenceis incorporated at the 3′-end of the first strand of the siNA moleculeand a nucleotide sequence that is complementary to the cellular targetnucleic acid sequence is incorporated at the 3′-end of the second strandof the siNA molecule (e.g., FIG. 1A). Alternately, the nucleotidesequence that is complementary to the HIV target nucleic acid sequenceis incorporated at the 5′-end of the first strand of the siNA moleculeand a nucleotide sequence that is complementary to the cellular targetnucleic acid sequence is incorporated at the 5′-end of the second strandof the siNA molecule (e.g., FIG. 1B). If self complementary, palindromeor repeat sequences are used, then generally, the longer the repeatidentified in the target nucleic acid sequence, the shorter theresulting siNA sequence will be. For example, if each target sequence is21 nucleotides in length and there is no repeat found in the sequence,the resulting siNA construct will be, for example, 21+0+21=42nucleotides in length. The first 21 nucleotides represent sequencecomplementary to the first target nucleic acid sequence, the 0represents the lack of a self complementary, palindrome, or repeatsequence, and the second 21 nucleotides represent sequence complementaryto the second target nucleic acid sequence. If a 2 nucleotide repeat isutilized, the resulting siNA construct will be, for example, 19+2+19=40nucleotides in length. If a 4 nucleotide repeat is utilized, theresulting siNA construct will be, for example, 17+4+17=38 nucleotides inlength. If a 6 nucleotide repeat is utilized, the resulting siNAconstruct will be, for example, 15+6+15=36 nucleotides in length. If an8 nucleotide repeat is utilized, the resulting siNA construct will be,for example, 13+8+13=34 nucleotides in length. If a 10 nucleotide repeatis utilized, the resulting siNA construct will be, for example,11+10+11=32 nucleotides in length. If a 12 nucleotide repeat isutilized, the resulting siNA construct will be, for example, 9+12+9=30nucleotides in length and so forth. Thus, for each nucleotide reductionin the target site, the siNA length can be shortened by 2 nucleotides.These same principles can be utilized for a target site having differentlength nucleotide sequences, such as target sites comprising 14 to 28nucleotides. In addition, various combinations of 5′ and 3′ overhangsequences (e.g., TT) can be introduced to the siNA constructs designedwith self complementary, palindrome, or repeat sequences.

In one example, multifunctional siNA are designed against HIV andcellular targets and are screened in cell culture experiments along withchemically modified siNA constructs with known activity using matchedchemistry inverted controls and untreated cells along with a trasfectioncontrol (LF2K). The siNA constructs of the invention can be used invarious cell culture systems as are commonly known in the art to screenfor compounds having anti-HIV activity. B cell, T cell, macrophage andendothelial cell culture systems are non-limiting examples of cellculture systems that can be readily adapted for screening siNA moleculesof the invention. In a non-limiting example, siNA molecules of theinvention are co-transfected with HIV-1 pNL4-3 proviral DNA into 293/EcRcells as described by Lee et al., 2002, Nature Biotechnology, 19,500-505, using a U6 snRNA promoter driven expression system.

In a non-limiting example, the siNA expression vectors are preparedusing the pTZ U6+1 vector described in Lee et al. supra. as follows. Onecassette harbors the first siNA strand and the other the second siNAstrand. These sequences are designed to target HIV and cellular RNAtargets described herein. As a control to verify a siNA mechanism,irrelevant sequences lacking complementarity to HIV and cellular targetsare subcloned in pTZ U6+1. RNA samples are prepared from 293/EcR cellstransiently co-transfected with siNA or control constructs, andsubjected to Ponasterone A induction. RNAs are also prepared from 293cells co-transfected with HIV-1 pNL4-3 proviral DNA and siNA or controlconstructs. For determination of anti-HIV activity of the siNAs,transient assays are done by co-transfection of siNA constructs andinfectious HIV proviral DNA, pNL4-3 into 293 cells as described above,followed by Northern analysis as known in the art. The p24 values arecalculated with the aid of, for example, a Dynatech MR5000 ELISA platereader (Dynatech Labs Inc., Chantilly, Va.). Cell viability can also beassessed using a Trypan Blue dye exclusion count at four days aftertransfection.

Other cell culture model systems for screening compounds having anti-HIVactivity are generally known in the art. For example, Duzgunes et al.,2001, Nucleosides, Nucleotides & Nucleic Acids, 20(4-7), 515-523; Cagnunet al., 2000, Antisense Nucleic Acid Drug Dev., 10, 251; Ho et al.,1995, Stem Cells, 13 supp 3, 100; and Baur et al., 1997, Blood, 89, 2259describe cell culture systems that can be readily adapted for use withboth synthetic and vector expressed multifunctional siNA compositions ofthe instant invention and the assays described herein.

Example 12 Animal Models

Various animal models can be used to screen multifunctional siNAconstructs in vivo as are known in the art, for example those animalmodels that are used to evaluate other nucleic acid technologies such asenzymatic nucleic acid molecules (ribozymes) and/or antisense. Suchanimal models are used to test the efficacy of siNA molecules describedherein. In a non-limiting example, siNA molecules that are designed asanti-angiogenic agents can be screened using animal models. There areseveral animal models available in which to test the anti-angiogenesiseffect of nucleic acids of the present invention, such as siNA, directedagainst genes associated with angiogenesis and/or metastasis, such asVEGF and VEGFR (e.g., VEGFR1, VEGFR2, and/or VEGFR3) or variouscombinations of VEGFR (e.g., VEGFR1, VEGFR2, and/or VEGFR3) genes.

Several animal models exist for screening of anti-angiogenic agents.These include corneal vessel formation following corneal injury (Burgeret al., 1985 Cornea 4: 35-41; Lepri, et al., 1994 J. Ocular Pharmacol.10: 273-280; Ormerod et al., 1990 Am. J. Pathol. 137: 1243-1252) orintracorneal growth factor implant (Grant et al., 1993 Diabetologia 36:282-291; Pandey et al. 1995 supra; Zieche et al., 1992 Lab. Invest. 67:711-715), vessel growth into Matrigel matrix containing growth factors(Passaniti et al., 1992 supra), female reproductive organneovascularization following hormonal manipulation (Shweiki et al., 1993Clin. Invest. 91: 2235-2243), several models involving inhibition oftumor growth in highly vascularized solid tumors (O'Reilly et al., 1994Cell 79: 315-328; Senger et al., 1993 Cancer and Metas. Rev. 12:303-324; Takahasi et al., 1994 Cancer Res. 54: 4233-4237; Kim et al.,1993 supra), and transient hypoxia-induced neovascularization in themouse retina (Pierce et al., 1995 Proc. Natl. Acad. Sci. USA. 92:905-909).

Ocular Models of Angiogenesis

The cornea model, described in Pandey et al. supra, is the most commonand well characterized model for screening anti-angiogenic agentefficacy. This model involves an avascular tissue into which vessels arerecruited by a stimulating agent (growth factor, thermal or alkali burn,endotoxin). The corneal model utilizes the intrastromal cornealimplantation of a Teflon pellet soaked in a VEGF-Hydron solution torecruit blood vessels toward the pellet, which can be quantitated usingstandard microscopic and image analysis techniques. To evaluate theiranti-angiogenic efficacy, nucleic acids are applied topically to the eyeor bound within Hydron on the Teflon pellet itself. This avascularcornea as well as the Matrigel (see below) provide for low backgroundassays. While the corneal model has been performed extensively in therabbit, studies in the rat have also been conducted.

The mouse model (Passaniti et al., supra) is a non-tissue model thatutilizes Matrigel, an extract of basement membrane (Kleinman et al.,1986) or Millipore® filter disk, which can be impregnated with growthfactors and anti-angiogenic agents in a liquid form prior to injection.Upon subcutaneous administration at body temperature, the Matrigel orMillipore® filter disk forms a solid implant. VEGF embedded in theMatrigel or Millipore® filter disk is used to recruit vessels within thematrix of the Matrigel or Millipore® filter disk which can be processedhistologically for endothelial cell specific vWF (factor VIII antigen)immunohistochemistry, Trichrome-Masson stain, or hemoglobin content.Like the cornea, the Matrigel or Millipore® filter disk is avascular;however, it is not tissue. In the Matrigel or Millipore® filter diskmodel, nucleic acids are administered within the matrix of the Matrigelor Millipore® filter disk to test their anti-angiogenic efficacy. Thus,delivery issues in this model, as with delivery of nucleic acids byHydron-coated Teflon pellets in the rat cornea model, may be lessproblematic due to the homogeneous presence of the nucleic acid withinthe respective matrix.

Additionally, siNA molecules of the invention targeting VEGF and/orVEGFR (e.g. VEGFR1, VEGFR2, and/or VEGFR3) can be assessed for activitytransgenic mice to determine whether modulation of VEGF and/or VEGFR caninhibit optic neovascularization. Animal models of choroidalneovascularization are described in, for example, Mori et al., 2001,Journal of Cellular Physiology, 188, 253; Mori et al., 2001, AmericanJournal of Pathology, 159, 313; Ohno-Matsui et al., 2002, AmericanJournal of Pathology, 160, 711; and Kwak et al., 2000, InvestigativeOphthalmology & Visual Science, 41, 3158. VEGF plays a central role incausing retinal neovascularization. Increased expression of VEGFR2 inretinal photoreceptors of transgenic mice stimulates neovascularizationwithin the retina, and a blockade of VEGFR2 signaling has been shown toinhibit retinal choroidal neovascularization (CNV) (Mori et al., 2001,J. Cell. Physiol., 188, 253).

CNV is laser induced in, for example, adult C57BL/6 mice. The mice arealso given an intravenous, periocular or a subretinal injection of VEGFand/or VEGFr (e.g., VEGFR2) siNA in each eye. Intravitreous injectionsare made using a Harvard pump microinjection apparatus and pulled glassmicropipets. Then a micropipette is passed through the sclera justbehind the limbus into the vitreous cavity. The subretinal injectionsare made using a condensing lens system on a dissecting microscope. Thepipet tip is then passed through the sclera posterior to the limbus andpositioned above the retina. Five days after the injection of the vectorthe mice are anesthetized with ketamine hydrochloride (100 mg/kg bodyweight), 1% tropicamide is also used to dilate the pupil, and a diodelaser photocoagulation is used to rupture Bruch's membrane at threelocations in each eye. A slit lamp delivery system and a hand-held coverslide are used for laser photocoagulation. Burns are made in the 9, 12,and 3 o'clock positions 2-3 disc diameters from the optic nerve (Mori etal., supra).

The mice typically develop subretinal neovascularization due to theexpression of VEGF in photoreceptors beginning at prenatal day 7. Atprenatal day 21, the mice are anesthetized and perfused with 1 ml ofphosphate-buffered saline containing 50 mg/ml of fluorescein-labeleddextran. Then the eyes are removed and placed for 1 hour in a 10%phosphate-buffered formalin. The retinas are removed and examined byfluorescence microscopy (Mori et al., supra).

Fourteen days after the laser induced rupture of Bruch's membrane, theeyes that received intravitreous and subretinal injection of siNA areevaluated for smaller appearing areas of CNV, while control eyes areevaluated for large areas of CNV. The eyes that receive intravitreousinjections or a subretinal injection of siNA are also evaluated forfewer areas of neovascularization on the outer surface of the retina andpotential abortive sprouts from deep retinal capillaries that do notreach the retinal surface compared to eyes that did not receive aninjection of siNA.

Tumor Models of Angiogenesis Use of Murine Models

For a typical systemic study involving 10 mice (20 g each) per dosegroup, 5 doses (1, 3, 10, 30 and 100 mg/kg daily over 14 days continuousadministration), approximately 400 mg of siRNA, formulated in saline isused. A similar study in young adult rats (200 g) requires over 4 g.Parallel pharmacokinetic studies involve the use of similar quantitiesof siRNA further justifying the use of murine models.

Lewis Lung Carcinoma and B-16 Melanoma Murine Models

Identifying a common animal model for systemic efficacy testing ofnucleic acids is an efficient way of screening siNA for systemicefficacy.

The Lewis lung carcinoma and B-16 murine melanoma models are wellaccepted models of primary and metastatic cancer and are used forinitial screening of anti-cancer agents. These murine models are notdependent upon the use of immunodeficient mice, are relativelyinexpensive, and minimize housing concerns. Both the Lewis lung and B-16melanoma models involve subcutaneous implantation of approximately 106tumor cells from metastatically aggressive tumor cell lines (Lewis lunglines 3LL or D122, LLc-LN7; B-16-BL6 melanoma) in C57BL/6J mice.Alternatively, the Lewis lung model can be produced by the surgicalimplantation of tumor spheres (approximately 0.8 mm in diameter).Metastasis also can be modeled by injecting the tumor cells directlyintravenously. In the Lewis lung model, microscopic metastases can beobserved approximately 14 days following implantation with quantifiablemacroscopic metastatic tumors developing within 21-25 days. The B-16melanoma exhibits a similar time course with tumor neovascularizationbeginning 4 days following implantation. Since both primary andmetastatic tumors exist in these models after 21-25 days in the sameanimal, multiple measurements can be taken as indices of efficacy.Primary tumor volume and growth latency as well as the number of micro-and macroscopic metastatic lung foci or number of animals exhibitingmetastases can be quantitated. The percent increase in lifespan can alsobe measured. Thus, these models provide suitable primary efficacy assaysfor screening systemically administered siRNA nucleic acids and siRNAnucleic acid formulations.

In the Lewis lung and B-16 melanoma models, systemic pharmacotherapywith a wide variety of agents usually begins 1-7 days following tumorimplantation/inoculation with either continuous or multipleadministration regimens. Concurrent pharmacokinetic studies can beperformed to determine whether sufficient tissue levels of siRNA can beachieved for pharmacodynamic effect to be expected. Furthermore, primarytumors and secondary lung metastases can be removed and subjected to avariety of in vitro studies (i.e. target RNA reduction).

Models of Angiogenesis Related Kidney Disease

In addition, animal models are useful in screening compounds, e.g. siNAmolecules, for efficacy in treating renal failure, such as a result ofautosomal dominant polycystic kidney disease (ADPKD). The Han:SPRD ratmodel, mice with a targeted mutation in the Pkd2 gene and congenitalpolycystic kidney (cpk) mice, closely resemble human ADPKD and provideanimal models to evaluate the therapeutic effect of siNA constructs thathave the potential to interfere with one or more of the pathogenicelements of ADPKD mediated renal failure, such as angiogenesis.Angiogenesis may be necessary in the progression of ADPKD for growth ofcyst cells as well as increased vascular permeability promoting fluidsecretion into cysts. Proliferation of cystic epithelium is also afeature of ADPKD because cyst cells in culture produce soluble vascularendothelial growth factor (VEGF). VEGFr1 has also been detected inepithelial cells of cystic tubules but not in endothelial cells in thevasculature of cystic kidneys or normal kidneys. VEGFr2 expression isincreased in endothelial cells of cyst vessels and in endothelial cellsduring renal ischemia-reperfusion. It is proposed that inhibition ofVEGF receptors with anti-VEGFr1 and anti-VEGFr2 siNA molecules wouldattenuate cyst formation, renal failure and mortality in ADPKD.Anti-VEGFr2 siNA molecules would therefore be designed to inhibitangiogenesis involved in cyst formation. As VEGFr1 is present in cysticepithelium and not in vascular endothelium of cysts, it is proposed thatanti-VEGFr1 siNA molecules would attenuate cystic epithelial cellproliferation and apoptosis which would in turn lead to less cystformation. Further, it is proposed that VEGF produced by cysticepithelial cells is one of the stimuli for angiogenesis as well asepithelial cell proliferation and apoptosis. The use of Han:SPRD rats(see for example Kaspareit-Rittinghausen et al., 1991, Am. J. Pathol.139, 693-696), mice with a targeted mutation in the Pkd2 gene (Pkd2−/−mice, see for example Wu et al., 2000, Nat. Genet. 24, 75-78) and cpkmice (see for example Woo et al., 1994, Nature, 368, 750-753) allprovide animal models to study the efficacy of siNA molecules of theinvention against VEGFr1 and VEGFr2 mediated renal failure.

VEGF, VEGFr1 VGFR2 and/or VEGFr3 protein levels can be measuredclinically or experimentally by FACS analysis. VEGF, VEGFr1 VGFR2 and/orVEGFr3 encoded mRNA levels are assessed by Northern analysis,RNase-protection, primer extension analysis and/or quantitative RT-PCR.siNA nucleic acids that block VEGF, VEGFr1 VGFR2 and/or VEGFr3 proteinencoding mRNAs and therefore result in decreased levels of VEGF, VEGFr1VGFR2 and/or VEGFr3 activity by more than 20% in vitro can beidentified.

TGF-Beta and TGF-Beta Receptor Animal Models

Evaluating the efficacy of anti-TGF-beta and/or TGF-betaR agents inanimal models is an important prerequisite to human clinical trials. Thefollowing description provides animal models for non-limiting examplesof diseases and conditions contemplated by the instant invention.

Diabetic Nephropathy:

The db/db mouse, which expresses a mutant form of the full length leptinreceptor in the hypothalamus, is a genetic model of type 2 diabetes thatdevelops hyperglycemia in the second month of age and overt nephropathyby four months of age. Additional animal models include thestreptozotocin diabetic rat or mouse, the spontaneously diabeticBioBreeding rat, and the nonobese diabetic mouse. These models areuseful in evaluating nucleic acid molecules of the invention targetingTGF-beta and TGF-betaR for efficacy in treating diabetic nephropathy.

Chronic Liver Disease:

The carbon tetrachloride-induced cirrhosis model in mice or rats is auseful model in studying chronic liver disease. In the mouse model,standard therapeutic regimens begin at week 12 and continue for at least10 weeks. Endpoints include serum chemistry (liver enzymes, directbilirubin), histopath evaluation with morphometric analysis of collagencontent, and liver hydroxyproline content. In the rat model, therapeuticregimens commence at week 6 and continue for up to week 16. Primaryendpoints are elevated liver enzyme profile and histopathologic evidenceof advanced fibrosis or frank cirrhosis. Phenobarbital can be added tothe induction regime and will up-regulate liver enzymes, allowing for afaster induction of the disease state. Liver panels are performed weeklyto monitor progression of the disease process. These models are usefulin evaluating nucleic acid molecules of the invention targeting TGF-betaand TGF-betaR for efficacy in treating chronic liver disease.

Pulmonary Fibrosis:

A rapid (14 day) bleomycin (Bleo)-induced pulmonary injury model isavailable in mice and in rats. This model is useful in evaluatingnucleic acid molecules of the invention targeting TGF-beta and TGF-betaRfor efficacy in treating pulmonary fibrosis.

HCV Animal Models

Evaluating the efficacy of anti-HCV agents in animal models is animportant prerequisite to human clinical trials. The best characterizedanimal system for HCV infection is the chimpanzee. Moreover, the chronichepatitis that results from HCV infection in chimpanzees and humans isvery similar. Although clinically relevant, the chimpanzee model suffersfrom several practical impediments that make use of this modeldifficult. These include high cost, long incubation requirements andlack of sufficient quantities of animals. Due to these factors, a numberof groups have attempted to develop rodent models of chronic hepatitis Cinfection. While direct infection has not been possible, several groupshave reported on the stable transfection of either portions or entireHCV genomes into rodents (Yamamoto et al., Hepatology 1995 22(3):847-855; Galun et al., Journal of Infectious Disease 1995 172(1):25-30;Koike et al., Journal of general Virology 1995 76(12)3031-3038;Pasquinelli et al., Hepatology 1997 25(3): 719-727; Hayashi et al.,Princess Takamatsu Symp 1995 25:1430149; Mariya et al., Journal ofGeneral Virology 1997 78(7) 1527-1531; Takehara et al., Hepatology 199521(3):746-751; Kawamura et al., Hepatology 1997 25(4): 1014-1021). Inaddition, transplantation of HCV infected human liver intoimmunocompromised mice results in prolonged detection of HCV RNA in theanimal's blood.

A method for expressing hepatitis C virus in an in vivo animal model hasbeen developed (Vierling, International PCT Publication No. WO99/16307). Viable, HCV infected human hepatocytes are transplanted intoa liver parenchyma of a scid/scid mouse host. The scid/scid mouse hostis then maintained in a viable state, whereby viable, morphologicallyintact human hepatocytes persist in the donor tissue and hepatitis Cvirus is replicated in the persisting human hepatocytes. This modelprovides an effective means for the study of HCV inhibition by enzymaticnucleic acids in vivo.

HIV Animal Models

Evaluating the efficacy of anti-HIV agents in animal models is animportant prerequisite to human clinical trials. The siNA constructs ofthe invention can be evaluated in a variety of animal models including,for example, a hollow fiber HIV model (see, for example, Gruenberg, U.S.Pat. No. 5,627,070), mouse models for AIDS using transgenic miceexpressing HIV-1 genes from CD4 promoters and enhancers (see, forexample, Jolicoeur, International PCT Publication No. WO 98/50535)and/or the HIV/SIV/SHIV non-human primate models (see, for example,Narayan, U.S. Pat. No. 5,849,994). The siNA compounds and virus can beadministered by a variety of methods and routes as described herein andas known in the art. Quantitation of results in these models can beperformed by a variety of methods, including quantitative PCR,quantitative and bulk co-cultivation assays, plasma co-cultivationassays, antigen and antibody detection assays, lymphocyte proliferation,intracellular cytokines, flow cytometry, as well as hematology and CBCevaluation. Additional animal models are generally known in the art, seefor example Bai et al., 2000, Mol. Ther., 1, 244.

HBV Animal Models

Non-limiting examples of HBV animal models useful in evaluating siNAmolecules of the invention are described in McSwiggen et al., U.S. Ser.No. 10/757,803 and U.S. Ser. No. 10/669,841, incorporated by referenceherein.

Example 10 Indications

The siNA molecules of the invention can be used to treat a variety ofdiseases and conditions through modulation of gene expression. Using themethods described herein, chemically modified siNA molecules can bedesigned to modulate the expression of any number of target genes,including but not limited to genes associated with cancer, metabolicdiseases, infectious diseases such as viral, bacterial or fungalinfections, neurologic diseases, musculoskeletal diseases, diseases ofthe immune system, diseases associated with signaling pathways andcellular messengers, and diseases associated with transport systemsincluding molecular pumps and channels.

Non-limiting examples of various viral genes that can be targeted usingsiNA molecules of the invention include Hepatitis C Virus (HCV, forexample Genbank Accession Nos: D11168, D50483.1, L38318 and S82227),Hepatitis B Virus (HBV, for example GenBank Accession No. AF100308.1),Human Immunodeficiency Virus type 1 (HIV-1, for example GenBankAccession No. U51188), Human Immunodeficiency Virus type 2 (HIV-2, forexample GenBank Accession No. X60667), West Nile Virus (WNV for exampleGenBank accession No. NC_(—)001563), cytomegalovirus (CMV for exampleGenBank Accession No. NC_(—)001347), respiratory syncytial virus (RSVfor example GenBank Accession No. NC_(—)001781), influenza virus (forexample GenBank Accession No. AF037412, rhinovirus (for example, GenBankaccession numbers: D00239, X02316, X01087, L24917, M16248, K02121,X01087), papillomavirus (for example GenBank Accession No. NC₁₃ 001353),Herpes Simplex Virus (HSV for example GenBank Accession No.NC_(—)001345), and other viruses such as HTLV (for example GenBankAccession No. AJ430458) and SARS (for example GenBank Accession No.NC_(—)004718). Due to the high sequence variability of many viralgenomes, selection of siNA molecules for broad therapeutic applicationswould likely involve the conserved regions of the viral genome.Nonlimiting examples of conserved regions of the viral genomes includebut are not limited to 5′-Non Coding Regions (NCR), 3′-Non CodingRegions (NCR) LTR regions and/or internal ribosome entry sites (IRES).siNA molecules designed against conserved regions of various viralgenomes will enable efficient inhibition of viral replication in diversepatient populations and may ensure the effectiveness of the siNAmolecules against viral quasi species which evolve due to mutations inthe non-conserved regions of the viral genome.

Non-limiting examples of human genes that can be targeted using siNAmolecules of the invention using methods described herein include anyhuman RNA sequence, for example those commonly referred to by GenbankAccession Number. These RNA sequences can be used to design siNAmolecules that inhibit gene expression and therefore abrogate diseases,conditions, or infections associated with expression of those genes.Such non-limiting examples of human genes that can be targeted usingsiNA molecules of the invention include VEGF (for example GenBankAccession No. NM_(—)003376.3), VEGFr (VEGFR1 for example GenBankAccession No. XM₁₃ 067723, VEGFR2 for example GenBank Accession No.AF063658), HER1, HER2, HER3, and HER4 (for example Genbank AccessionNos: NM_(—)005228, NM_(—)004448, NM_(—)001982, and NM_(—)005235respectively), telomerase (TERT, for example GenBank Accession No.NM_(—)003219), telomerase RNA (for example GenBank Accession No.U86046), NFkappaB, Rel-A (for example GenBank Accession No.NM_(—)005228), NOGO (for example GenBank Accession No. AB020693), NOGOr(for example GenBank Accession No. XM_(—)015620), RAS (for exampleGenBank Accession No. NM₁₃ 004283), RAF (for example GenBank AccessionNo. XM_(—)033884), CD20 (for example GenBank Accession No. X07203),METAP2 (for example GenBank Accession No. NM₁₃ 003219), CLCA1 (forexample GenBank Accession No. NM_(—)001285), phospholamban (for exampleGenBank Accession No. NM_(—)002667), PTP1B (for example GenBankAccession No. M31724), PCNA (for example GenBank Accession No.NM_(—)002592.1), PKC-alpha (for example GenBank Accession No.NM_(—)002737) and others. The genes described herein are provided asnon-limiting examples of genes that can be targeted using siNA moleculesof the invention. Additional examples of such genes are described byaccession number in Beigelman et al., U.S. Ser. No. 60/363,124, filedMar. 11, 2002 and incorporated by reference herein in its entirety.

The siNA molecule of the invention can also be used in a variety ofagricultural applications involving modulation of endogenous orexogenous gene expression in plants using siNA, including use asinsecticidal, antiviral and anti-fungal agents or modulate plant traitssuch as oil and starch profiles and stress resistance.

Example 11 Diagnostic Uses

The siNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of siNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. siNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells or to detect the presence of endogenous orexogenous, for example viral, RNA in a cell. The close relationshipbetween siNA activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule, which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple siNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target RNAs withsiNA molecules can be used to inhibit gene expression and define therole of specified gene products in the progression of disease orinfection. In this manner, other genetic targets can be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes, siNA molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations siNA moleculesand/or other chemical or biological molecules). Other in vitro uses ofsiNA molecules of this invention are well known in the art, and includedetection of the presence of mRNAs associated with a disease, infection,or related condition. Such RNA is detected by determining the presenceof a cleavage product after treatment with a siNA using standardmethodologies, for example, fluorescence resonance emission transfer(FRET).

In a specific example, siNA molecules that cleave only wild-type ormutant forms of the target RNA are used for the assay. The first siNAmolecules (i.e., those that cleave only wild-type forms of target RNA)are used to identify wild-type RNA present in the sample and the secondsiNA molecules (i.e., those that cleave only mutant forms of target RNA)are used to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth siNA molecules to demonstrate the relative siNA efficiencies in thereactions and the absence of cleavage of the “non-targeted” RNA species.The cleavage products from the synthetic substrates also serve togenerate size markers for the analysis of wild-type and mutant RNAs inthe sample population. Thus, each analysis requires two siNA molecules,two substrates and one unknown sample, which is combined into sixreactions. The presence of cleavage products is determined using anRNase protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype (i.e., disease related orinfection related) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels is adequate and decreases the costof the initial diagnosis. Higher mutant form to wild-type ratios arecorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I Multifunctional siNA sequences Seq Seq Target1 Pos1Target1 Sequence ID Target1 Complement ID VEGFR1  349CUGAGUUUAAAAGGCACCC   8 GGGUGCCUUUUAAACUCAG  66 VEGFR1 1175AUACUUGUCGUGUAAGGAG   9 CUCCUUACACGACAAGUAU  67 VEGFR1 1501CUCACUGCCACUCUAAUUG  10 CAAUUAGAGUGGCAGUGAG  68 VEGFR1 1502UCACUGCCACUCUAAUUGU  11 ACAAUUAGAGUGGCAGUGA  69 VEGFR1 1503CACUGCCACUCUAAUUGUC  12 GACAAUUAGAGUGGCAGUG  70 VEGFR1 1504ACUGCCACUCUAAUUGUCA  13 UGACAAUUAGAGUGGCAGU  71 VEGFR1 3646AUGCUGGACUGCUGGCACA  14 UGUGCCAGCAGUCCAGCAU  72 Seq Seq Target2  Pos2Target2 Sequence ID Target2 Complement ID VEGFR2 3715ACCAUGCUGGACUGCUGGC  26 GCCAGCAGUCCAGCAUGGU  84 VEGFR2 2332ACGACAAGUAUUGGGGAAA 121 UUUCCCCAAUACUUGUCGU 142 VEGFR2  503AGAGUGGCAGUGAGCAAAG 122 CUUUGCUCACUGCCACUCU 143 VEGFR2  503AGAGUGGCAGUGAGCAAAG 122 CUUUGCUCACUGCCACUCU 143 VEGFR2  503AGAGUGGCAGUGAGCAAAG 122 CUUUGCUCACUGCCACUCU 143 VEGFR2  503AGAGUGGCAGUGAGCAAAG 122 CUUUGCUCACUGCCACUCU 143 VEGFR2 3716CCAUGCUGGACUGCUGGCA 27 UGCCAGCAGUCCAGCAUGG  85 Seq SeqMultifunctional Strand 1 (VEGFR2) ID Multifunctional Strand 2 (VEGFR1)ID UCAUCAUUAAUUUUUGCUUGCCAUUCCC 180 GGGAAUGGCAAGCAAAAAUUAAUGAUGA 232AGUGGAUGUGAUGCGGGGGCUGCUGCAA 181 UUGCAGCAGCCCCCGCAUCACAUCCACU 233ACAUGAUCUGUGGAGGGGGUCGGGGCUC 182 GAGCCCCGACCCCCUCCACAGAUCAUGU 234CCUGCAAGUUGCUGUCUUGGGUGCAUUG 183 CAAUGCACCCAAGACAGCAACUUGCAGG 235GCCAGCAGUCCAGCAUGGUUGGACAUCUUCCAGGAGUA 184UACUCCUGGAAGAUGUCCAACCAUGCUGGACUGCUGGC 236UGCCAGCAGUCCAGCAUGGUGUGAAUGCAGACCAAAGA 185UCUUUGGUCUGCAUUCACACCAUGCUGGACUGCUGGCA 237GUGCCAGCAGUCCAGCAUGGUGAAUGCAGACCAAAGAA 186UUCUUUGGUCUGCAUUCACCAUGCUGGACUGCUGGCAC 238 CACAGACUCCCUGCUUUUGGGGGUGACC187 GGUCACCCCCAAAAGCAGGGAGUCUGUG 239

TABLE II Seq Seq Target1 Pos1 Target1 Sequence ID Target1 Complement IDVEGF  143 CAUUGAUCCGGGUUUUAUC  41 GAUAAAACCCGGAUCAAUG  99 VEGF  181CAUUUUUUUUUAAAACUGU  42 ACAGUUUUAAAAAAAAAUG 100 VEGF  221UUUUUGCUUGCCAUUCCCC  43 GGGGAAUGGCAAGCAAAAA 101 VEGF  360AGAGACGGGGUCAGAGAGA  44 UCUCUCUGACCCCGUCUCU 102 VEGF  361GAGACGGGGUCAGAGAGAG  45 CUCUCUCUGACCCCGUCUC 103 VEGF  598CCGGAGCCCGCGCCCGGAG  46 CUCCGGGCGCGGGCUCCGG 104 VEGF  758GGGAGGAGCCGCAGCCGGA  47 UCCGGCUGCGGCUCCUCCC 105 VEGF 1062GCAUUGGAGCCUUGCCUUG  48 CAAGGCAAGGCUCCAAUGC 106 VEGF 1214UGGACAUCUUCCAGGAGUA  49 UACUCCUGGAAGAUGUCCA 107 VEGF 1420UGUGAAUGCAGACCAAAGA  50 UCUUUGGUCUGCAUUCACA 108 VEGF 1421GUGAAUGCAGACCAAAGAA  51 UUCUUUGGUCUGCAUUCAC 109 VEGF 1562AGCAUUUGUUUGUACAAGA  52 UCUUGUACAAACAAAUGCU 110 VEGF 1563GCAUUUGUUUGUACAAGAU  53 AUCUUGUACAAACAAAUGC 111 Seq Seq Target2 Pos2Target2 Sequence ID Target2 Complement ID VEGFR1 3646AUGCUGGACUGCUGGCACA  14 UGUGCCAGCAGUCCAGCAU  72 VEGFR1 3646AUGCUGGACUGCUGGCACA  14 UGUGCCAGCAGUCCAGCAU  72 VEGFR1 2063GCAAGCAAAAAAUGGCCAU 134 AUGGCCAUUUUUUGCUUGC 155 VEGFR1 5353GACCCCGUCUCUAUACCAA 135 UUGGUAUAGAGACGGGGUC 156 VEGFR1 5353GACCCCGUCUCUAUACCAA 135 UUGGUAUAGAGACGGGGUC 156 VEGFR1   47GCGGGCUCCGGGGCUCGGG 136 CCCGAGCCCCGGAGCCCGC 157 VEGFR1   15CGGCUCCUCCCCGGCAGCG 137 CGCUGCCGGGGAGGAGCCG 158 VEGFR1 3646AUGCUGGACUGCUGGCACA  14 UGUGCCAGCAGUCCAGCAU  72 VEGFR1  349CUGAGUUUAAAAGGCACCC   8 GGGUGCCUUUUAAACUCAG  66 VEGFR1 3646AUGCUGGACUGCUGGCACA  14 UGUGCCAGCAGUCCAGCAU  72 VEGFR1  349CUGAGUUUAAAAGGCACCC   8 GGGUGCCUUUUAAACUCAG  66 VEGFR1 3646AUGCUGGACUGCUGGCACA  14 UGUGCCAGCAGUCCAGCAU  72 VEGFR1 3646AUGCUGGACUGCUGGCACA  14 UGUGCCAGCAGUCCAGCAU  72 Seq SeqMultifunctional Strand 1 (VEGFR1) ID Multifunctional Strand 2 (VEGF)  IDUGUGCCAGCAGUCCAGCAUUGAUCCGGGUUUUAUC 199GAUAAAACCCGGAUCAAUGCUGGACUGCUGGCACA 249UGUGCCAGCAGUCCAGCAUUUUUUUUUAAAACUGU 200ACAGUUUUAAAAAAAAAUGCUGGACUGCUGGCACA 250 GGGGAAUGGCAAGCAAAAAAUGGCCAU 201AUGGCCAUUUUUUGCUUGCCAUUCCCC 251 UCUCUCUGACCCCGUCUCUAUACCAA 202UUGGUAUAGAGACGGGGUCAGAGAGA 252 CUCUCUCUGACCCCGUCUCUAUACCAA 203UUGGUAUAGAGACGGGGUCAGAGAGAG 253 CUCCGGGCGCGGGCUCCGGGGCUCGGG 204CCCGAGCCCCGGAGCCCGCGCCCGGAG 254 UCCGGCUGCGGCUCCUCCCCGGCAGCG 205CGCUGCCGGGGAGGAGCCGCAGCCGGA 255 UGUGCCAGCAGUCCAGCAUUGGAGCCUUGCCUUG 206CAAGGCAAGGCUCCAAUGCUGGACUGCUGGCACA 256UACUCCUGGAAGAUGUCCACUGAGUUUAAAAGGCACCC 207GGGUGCCUUUUAAACUCAGUGGACAUCUUCCAGGAGUA 257UCUUUGGUCUGCAUUCACAAUGCUGGACUGCUGGCACA 208UGUGCCAGCAGUCCAGCAUUGUGAAUGCAGACCAAAGA 258UUCUUUGGUCUGCAUUCACCUGAGUUUAAAAGGCACCC 209GGGUGCCUUUUAAACUCAGGUGAAUGCAGACCAAAGAA 259UGUGCCAGCAGUCCAGCAUUUGUUUGUACAAGA 210 UCUUGUACAAACAAAUGCUGGACUGCUGGCACA260 UGUGCCAGCAGUCCAGCAUUUGUUUGUACAAGAU 211AUCUUGUACAAACAAAUGCUGGACUGCUGGCACA 261

TABLE III Seq Seq Target1 Pos1 Target1 Sequence ID Target1 Complement IDVEGF  851 CAUGGACGGGUGAGGCGGC  54 GCCGCCUCACCCGUCCAUG 112 VEGF  852AUGGACGGGUGAGGCGGCG  55 CGCCGCCUCACCCGUCCAU 113 VEGF 1122CAUGGCAGAAGGAGGAGGG  56 CCCUCCUCCUUCUGCCAUG 114 VEGF 1123AUGGCAGAAGGAGGAGGGC  57 GCCCUCCUCCUUCUGCCAU 115 VEGF 1167CAUGGAUGUCUAUCAGCGC  58 GCGCUGAUAGACAUCCAUG 116 Seq Seq Target2  Pos2Target2 Sequence ID Target2 Complement ID VEGFR2 3716CCAUGCUGGACUGCUGGCA  27 UGCCAGCAGUCCAGCAUGG  85 VEGFR2 3716CCAUGCUGGACUGCUGGCA  27 UGCCAGCAGUCCAGCAUGG  85 VEGFR2 3716CCAUGCUGGACUGCUGGCA  27 UGCCAGCAGUCCAGCAUGG  85 VEGFR2 3716CCAUGCUGGACUGCUGGCA  27 UGCCAGCAGUCCAGCAUGG  85 VEGFR2 3716CCAUGCUGGACUGCUGGCA  27 UGCCAGCAGUCCAGCAUGG  85 Seq SeqMultifunctional Strand 1 (VEGFR2) ID Multifunctional Strand 2 (VEGF) IDUGCCAGCAGUCCAGCAUGGACGGGUGAGGCGGC 212 GCCGCCUCACCCGUCCAUGCUGGACUGCUGGCA262 UGCCAGCAGUCCAGCAUGGACGGGUGAGGCGGCG 213CGCCGCCUCACCCGUCCAUGCUGGACUGCUGGCA 263 UGCCAGCAGUCCAGCAUGGCAGAAGGAGGAGGG214 CCCUCCUCCUUCUGCCAUGCUGGACUGCUGGCA 264UGCCAGCAGUCCAGCAUGGCAGAAGGAGGAGGGC 215GCCCUCCUCCUUCUGCCAUGCUGGACUGCUGGCA 265 UGCCAGCAGUCCAGCAUGGAUGUCUAUCAGCGC216 GCGCUGAUAGACAUCCAUGCUGGACUGCUGGCA 266

TABLE IV Seq Seq Target1 Pos1 Target1 Sequence ID Target1 Complement IDVEGF  143 CAUUGAUCCGGGUUUUAUC  41 GAUAAAACCCGGAUCAAUG  99 VEGF  181CAUUUUUUUUUAAAACUGU  42 ACAGUUUUAAAAAAAAAUG 100 VEGF 1062GCAUUGGAGCCUUGCCUUG  48 CAAGGCAAGGCUCCAAUGC 106 VEGF 1420UGUGAAUGCAGACCAAAGA  50 UCUUUGGUCUGCAUUCACA 108 VEGF 1562AGCAUUUGUUUGUACAAGA  52 UCUUGUACAAACAAAUGCU 110 VEGF 1563GCAUUUGUUUGUACAAGAU  53 AUCUUGUACAAACAAAUGC 111 VEGF  851CAUGGACGGGUGAGGCGGC  54 GCCGCCUCACCCGUCCAUG 112 VEGF  852AUGGACGGGUGAGGCGGCG  55 CGCCGCCUCACCCGUCCAU 113 VEGF 1122CAUGGCAGAAGGAGGAGGG  56 CCCUCCUCCUUCUGCCAUG 114 VEGF 1123AUGGCAGAAGGAGGAGGGC  57 GCCCUCCUCCUUCUGCCAU 115 VEGF 1167CAUGGAUGUCUAUCAGCGC  58 GCGCUGAUAGACAUCCAUG 116 Seq Seq Target2 Pos2Target2 Sequence ID Target2 Complement ID VEGFR1/R2 3646AUGCUGGACUGCUGGCACA  14 UGUGCCAGCAGUCCAGCAU  72 VEGFR1/R2 3646AUGCUGGACUGCUGGCACA  14 UGUGCCAGCAGUCCAGCAU  72 VEGFR1/R2 3646AUGCUGGACUGCUGGCACA  14 UGUGCCAGCAGUCCAGCAU  72 VEGFR1/R2 3646AUGCUGGACUGCUGGCACA  14 UGUGCCAGCAGUCCAGCAU  72 VEGFR1/R2 3646AUGCUGGACUGCUGGCACA  14 UGUGCCAGCAGUCCAGCAU  72 VEGFR1/R2 3646AUGCUGGACUGCUGGCACA  14 UGUGCCAGCAGUCCAGCAU  72 VEGFR1/R2 3716CCAUGCUGGACUGCUGGCA  27 UGCCAGCAGUCCAGCAUGG  85 VEGFR1/R2 3716CCAUGCUGGACUGCUGGCA  27 UGCCAGCAGUCCAGCAUGG  85 VEGFR1/R2 3716CCAUGCUGGACUGCUGGCA  27 UGCCAGCAGUCCAGCAUGG  85 VEGFR1/R2 3716CCAUGCUGGACUGCUGGCA  27 UGCCAGCAGUCCAGCAUGG  85 VEGFR1/R2 3716CCAUGCUGGACUGCUGGCA  27 UGCCAGCAGUCCAGCAUGG  85 Seq SeqMultifunctional Strand 1 (VEGFR1/R2) ID Multifunctional Strand 2 (VEGF)ID UGUGCCAGCAGUCCAGCAUUGAUCCGGGUUUUAUC 199GAUAAAACCCGGAUCAAUGCUGGACUGCUGGCACA 249UGUGCCAGCAGUCCAGCAUUUUUUUUUAAAACUGU 200ACAGUUUUAAAAAAAAAUGCUGGACUGCUGGCACA 250UGUGCCAGCAGUCCAGCAUUGGAGCCUUGCCUUG 206CAAGGCAAGGCUCCAAUGCUGGACUGCUGGCACA 256UCUUUGGUCUGCAUUCACAAUGCUGGACUGCUGGCACA 208UGUGCCAGCAGUCCAGCAUUGUGAAUGCAGACCAAAGA 258UGUGCCAGCAGUCCAGCAUUUGUUUGUACAAGA 210 UCUUGUACAAACAAAUGCUGGACUGCUGGCACA260 UGUGCCAGCAGUCCAGCAUUUGUUUGUACAAGAU 211AUCUUGUACAAACAAAUGCUGGACUGCUGGCACA 261 UGCCAGCAGUCCAGCAUGGACGGGUGAGGCGGC212 GCCGCCUCACCCGUCCAUGCUGGACUGCUGGCA 262UGCCAGCAGUCCAGCAUGGACGGGUGAGGCGGCG 213CGCCGCCUCACCCGUCCAUGCUGGACUGCUGGCA 263 UGCCAGCAGUCCAGCAUGGCAGAAGGAGGAGGG214 CCCUCCUCCUUCUGCCAUGCUGGACUGCUGGCA 264UGCCAGCAGUCCAGCAUGGCAGAAGGAGGAGGGC 215GCCCUCCUCCUUCUGCCAUGCUGGACUGCUGGCA 265 UGCCAGCAGUCCAGCAUGGAUGUCUAUCAGCGC216 GCGCUGAUAGACAUCCAUGCUGGACUGCUGGCA 266

TABLE V Seq Seq Target1 Pos1 Target1 Sequence ID Target1 Complement IDHCV  141 CGGGAGAGCCAUAGUGGUC  15 GACCACUAUGGCUCUCCCG  73 HCV  151AUAGUGGUCUGCGGAACCG  16 CGGUUCCGCAGACCACUAU  74 HCV  152UAGUGGUCUGCGGAACCGG  17 CCGGUUCCGCAGACCACUA  75 HCV  287AGGCCUUGUGGUACUGCCU  18 AGGCAGUACCACAAGGCCU  76 HCV  300CUGCCUGAUAGGGUGCUUG  19 CAAGCACCCUAUCAGGCAG  77 HCV  304CUGAUAGGGUGCUUGCGAG  20 CUCGCAAGCACCCUAUCAG  78 HCV  327CCGGGAGGUCUCGUAGACC  21 GGUCUACGAGACCUCCCGG  79 Seq Seq Target2 Pos2Target2 Sequence ID Target2 Complement ID FAS  272 UCUCCCGCGGGUUGGUGGA  3 UCCACCAACCCGCGGGAGA  61 FAS 1046 ACCACUAUUGCUGGAGUCA   5UGACUCCAGCAAUAGUGGU  63 FAS 1046 ACCACUAUUGCUGGAGUCA   5UGACUCCAGCAAUAGUGGU  63 FAS  495 AAGGCCUGCAUCAUGAUGG   4CCAUCAUGAUGCAGGCCUU  62 FAS 1771 CAGGCAGGCCACUUUGCCU 123AGGCAAAGUGGCCUGCCUG 144 FAS 1757 UAUCAGUUACUGAACAGGC 124GCCUGUUCAGUAACUGAUA 145 FAS   49 CCCGGGCGUUCCCCAGCGA 125UCGCUGGGGAACGCCCGGG 146 Seq Seq Multifunctional Strand 1 (FAS) IDMultifunctional Strand 2 (HCV) ID GACCACUAUGGCUCUCCCGCGGGUUGGUGGA 173UCCACCAACCCGCGGGAGAGCCAUAGUGGUC 161 CGGUUCCGCAGACCACUAUUGCUGGAGUCA 174UGACUCCAGCAAUAGUGGUCUGCGGAACCG 163 CCGGUUCCGCAGACCACUAUUGCUGGAGUCA 175UGACUCCAGCAAUAGUGGUCUGCGGAACCGG 228 AGGCAGUACCACAAGGCCUGCAUCAUGAUGG 176CCAUCAUGAUGCAGGCCUUGUGGUACUGCCU 162 CAAGCACCCUAUCAGGCAGGCCACUUUGCCU 177AGGCAAAGUGGCCUGCCUGAUAGGGUGCUUG 229 CUCGCAAGCACCCUAUCAGUUACUGAACAGGC 178GCCUGUUCAGUAACUGAUAGGGUGCUUGCGAG 230 GGUCUACGAGACCUCCCGGGCGUUCCCCAGCGA179 UCGCUGGGGAACGCCCGGGAGGUCUCGUAGACC 231

TABLE VI Seq Seq Target1 Pos1 Target1 Sequence ID Target1 Complement IDTGFB1   10 CGCGGAGCAGCCAGACAGC  30 GCUGUCUGGCUGCUCCGCG  88 TGFB1  135GAGGAGCAGCCUGAGGCCC  31 GGGCCUCAGGCUGCUCCUC  89 TGFB1  169GCCGCCGCCGCCCCCGCCA  32 UGGCGGGGGCGGCGGCGGC  90 TGFB1  170CCGCCGCCGCCCCCGCCAC  33 GUGGCGGGGGCGGCGGCGG  91 TGFB1  364GCCGGGGACGCUUGCUCCC  34 GGGAGCAAGCGUCCCCGGC  92 TGFB1 1528GGAUAACACACUGCAAGUG  35 CACUUGCAGUGUGUUAUCC  93 TGFB1 2385AUAGCAACACUCUGAGAUG  36 CAUCUCAGAGUGUUGCUAU  94 Seq Seq Target2 Pos2Target2 Sequence ID Target2 Complement ID TGFBR1   92GCUGCUCCGCGUCCCCGGC 131 GCCGGGGACGCGGAGCAGC 152 TGFBR1  108GGCUGCUCCUCCUCGUGCU 132 AGCACGAGGAGGAGCAGCC 153 TGFBR1  127GGCGGCGGCGGCGGCGGCG  37 CGCCGCCGCCGCCGCCGCC  95 TGFBR1  127GGCGGCGGCGGCGGCGGCG  37 CGCCGCCGCCGCCGCCGCC  95 TGFBR1  100GCGUCCCCGGCUGCUCCUC 133 GAGGAGCAGCCGGGGACGC 154 TGFBR1 1770GGGUCCUUUCUGUGCACUA  40 UAGUGCACAGAAAGGACCC  98 TGFBR1 1565CAACAGGAAGGCAUCAAAA  39 UUUUGAUGCCUUCCUGUUG  97 Seq SeqMultifunctional Strand 1 (TGFBR1) ID Multifunctional Strand 2 (TGFB1) IDGCUGUCUGGCUGCUCCGCGUCCCCGGC 188 GCCGGGGACGCGGAGCAGCCAGACAGC 240GGGCCUCAGGCUGCUCCUCCUCGUGCU 189 AGCACGAGGAGGAGCAGCCUGAGGCCC 241UGGCGGGGGCGGCGGCGGCGGCGGCG 190 CGCCGCCGCCGCCGCCGCCCCCGCCA 195GUGGCGGGGGCGGCGGCGGCGGCGGCG 191 CGCCGCCGCCGCCGCCGCCCCCGCCAC 242GGGAGCAAGCGUCCCCGGCUGCUCCUC 192 GAGGAGCAGCCGGGGACGCUUGCUCCC 243CACUUGCAGUGUGUUAUCCGGGUCCUUUCUGUGCACUA 193UAGUGCACAGAAAGGACCCGGAUAACACACUGCAAGUG 244CAUCUCAGAGUGUUGCUAUCAACAGGAAGGCAUCAAAA 194UUUUGAUGCCUUCCUGUUGAUAGCAACACUCUGAGAUG 245

TABLE VII Wait Time* Wait Time* Wait Time* Reagent Equivalents AmountDNA 2′-O-methyl RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL  5 sec 5sec 5 sec N-Methyl 186 233 μL  5 sec 5 sec 5 sec Imidazole TCA 176 2.3mL 21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec  300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 μL 45sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 secAcetic Anhydride 655 124 μL  5 sec 5 sec 5 sec N-Methyl 1245 124 μL  5sec 5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 sec Iodine20.6 244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec  300 sec300 sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96well Instrument Equivalents: DNA/ Amount: DNA/2′-O- Wait Time* WaitTime* Wait Time* Reagent 2′-O-methyl/Ribo methyl/Ribo DNA 2′-O-methylRibo Phosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 sec S-Ethyl Tetrazole 70/105/210 40/60/120 μL 60 sec 180 min 360 sec  AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec  200 sec 200 sec Acetonitrile NA 1150/1150/1150 μL NA NA NA •Wait time does not includecontact time during delivery. •Tandem synthesis utilizes double couplingof linker molecule

TABLE VIII Non-limiting examples of Stabilization Chemistries forchemically modified multifunctional siNA constructs Chemistry pyrimidinePurine cap p = S “Stab 00” Ribo Ribo TT at 3′-ends “Stab 1” Ribo Ribo —5 at 5′-end 1 at 3′-end “Stab 2” Ribo Ribo — All linkages “Stab 3”2′-fluoro Ribo — 4 at 5′-end 4 at 3′-end “Stab 4” 2′-fluoro Ribo 3′-end— “Stab 5” 2′-fluoro Ribo — 1 at 3′-end “Stab 6” 2′-O-Methyl Ribo 3′-end— “Stab 7” 2′-fluoro 2′-deoxy 3′-end — “Stab 8” 2′-fluoro 2′-O- — 1 at3′-end Methyl “Stab 9” Ribo Ribo 3′-end — “Stab 10” Ribo Ribo — 1 at3′-end “Stab 11” 2′-fluoro 2′-deoxy — 1 at 3′-end “Stab 12” 2′-fluoroLNA 3′-end “Stab 13” 2′-fluoro LNA 1 at 3′-end “Stab 14” 2′-fluoro2′-deoxy 2 at 5′-end 1 at 3′-end “Stab 15” 2′-deoxy 2′-deoxy 2 at 5′-end1 at 3′-end “Stab 16” Ribo 2′-O- 3′-end Methyl “Stab 17” 2′-O-Methyl2′-O- 3′-end Methyl “Stab 18” 2′-fluoro 2′-O- 3′-end 1 at 3′-end Methyl“Stab 19” 2′-fluoro 2′-O- 3′-end Methyl “Stab 20” 2′-fluoro 2′-deoxy3′-end “Stab 21” 2′-fluoro Ribo 3′-end “Stab 22” Ribo Ribo 3′-end CAP =any terminal cap, see for example FIG. 9. All Stab 1-22 chemistries cancomprise 3′-terminal thymidine (TT) residues

1. A multifunctional siNA molecule of Formula II: 5′-p-X X′-3′ 3′-Y′Y-p-5′

wherein each 5′-p-XX′-3′ and 5′-p-YY′-3′ independently comprise anoligonucleotide of length between about 24 and about 38 nucleotides, Xcomprises a nucleic acid sequence that is complementary to a firsttarget nucleic acid sequence, Y comprises an oligonucleotide comprisingnucleic acid sequence that is complementary to a second target nucleicacid sequence, said X further comprises nucleotide sequence of lengthabout 1 to about 21 nucleotides that is complementary to nucleotidesequence present in region Y′, said Y further comprises nucleotidesequence of length about 1 to about 21 nucleotides that is complementaryto nucleotide sequence present in region X′, p comprises a terminalphosphate group that can independently be present or absent, and whereineach said X and said Y are independently of length sufficient to stablyinteract with said first and said second target nucleic acid sequence,respectively, or a portion thereof.
 2. The siNA molecule of claim 1,wherein said siNA comprises a 3′-terminal cap moiety.
 3. The siNAmolecule of claim 2, wherein said terminal cap moiety is an inverteddeoxyabasic moiety.
 4. The siNA molecule of claim 2, wherein saidterminal cap moiety is an inverted deoxynucleotide moiety.
 5. The siNAmolecule of claim 2, wherein said terminal cap moiety is a dinucleotidemoiety.
 6. The siNA molecule of claim 5, wherein said dinucleotide isdithymidine (TT).
 7. The siNA molecule of claim 1, wherein said siNAmolecule comprises no ribonucleotides.
 8. The siNA molecule of claim 1,wherein said siNA molecule comprises ribonucleotides.
 9. The siNAmolecule of claim 1, wherein any purine nucleotide in said siNA is a2′-O-methylpyrimidine nucleotide.
 10. The siNA molecule of claim 1,wherein any purine nucleotide in said siNA is a 2′-deoxy purinenucleotide.
 11. The siNA molecule of claim 1, wherein any pyrimidinenucleotide in said siNA is a 2′-deoxy-2′-fluoro pyrimidine nucleotide.12. The siNA molecule of claim 1, wherein said siNA molecule comprises3′-nucleotide overhangs.
 13. The siNA molecule of claim 12, wherein said3′-overhangs comprise about 1 to about 4 nucleotides.
 14. The siNAmolecule of claim 13, wherein said nucleotides comprisedeoxynucleotides.
 15. The siNA molecule of claim 14, wherein saiddeoxynucleotides are thymidine nucleotides.
 16. A pharmaceuticalcomposition comprising the siNA molecule of claim 1 in an acceptablecarrier or diluent.